Substituted imidazopyridazines

ABSTRACT

The present invention relates to substituted imidazopyridazine compounds, to methods of preparing said compounds, to pharmaceutical compositions and combinations comprising said compounds and to the use of said compounds for manufacturing a pharmaceutical composition for the treatment or prophylaxis of a disease, in particular of a hyper-proliferative and/or angiogenesis disorder, as a sole agent or in combination with other active ingredients.

The present invention relates to substituted imidazopyridazine compoundsas described and defined herein, to methods of preparing said compounds,to pharmaceutical compositions and combinations comprising saidcompounds, to the use of said compounds for manufacturing apharmaceutical composition for the treatment or prophylaxis of adisease, as well as to intermediate compounds useful in the preparationof said compounds.

BACKGROUND OF THE INVENTION

The present invention relates to chemical compounds that inhibit Mps-1(Monopolar Spindle 1) kinase (also known as Tyrosine Threonine Kinase,TTK). Mps-1 is a dual specificity Ser/Thr kinase which plays a key rolein the activation of the mitotic checkpoint (also known as spindlecheckpoint, spindle assembly checkpoint) thereby ensuring properchromosome segregation during mitosis [Abrieu A et al., Cell, 2001, 106,83-93]. Every dividing cell has to ensure equal separation of thereplicated chromosomes into the two daughter cells. Upon entry intomitosis, chromosomes are attached at their kinetochores to themicrotubules of the spindle apparatus. The mitotic checkpoint is asurveillance mechanism that is active as long as unattached kinetochoresare present and prevents mitotic cells from entering anaphase andthereby completing cell division with unattached chromosomes[Suijkerbuijk S J and Kops G J, Biochemica et Biophysica Acta, 2008,1786, 24-31; Musacchio A and Salmon E D, Nat Rev Mot Cell Biol., 2007,8, 379-93]. Once all kinetochores are attached in a correct amphitelic,i.e. bipolar, fashion with the mitotic spindle, the checkpoint issatisfied and the cell enters anaphase and proceeds through mitosis. Themitotic checkpoint consists of complex network of a number of essentialproteins, including members of the MAD (mitotic arrest deficient, MAD1-3) and Bub (Budding uninhibited by benzimidazole, Bub 1-3) families,the motor protein CENP-E, Mps-1 kinase as well as other components, manyof these being over-expressed in proliferating cells (e.g. cancer cells)and tissues [Yuan B et al., Clinical Cancer Research, 2006, 12, 405-10].The essential role of Mps-1 kinase activity in mitotic checkpointsignalling has been shown by shRNA-silencing, chemical genetics as wellas chemical inhibitors of Mps-1 kinase [Jelluma N et al., PLos ONE,2008, 3, e2415; Jones M H et al., Current Biology, 2005, 15, 160-65;Dorer R K et al., Current Biology, 2005, 15, 1070-76; Schmidt M et al.,EMBO Reports, 2005, 6, 866-72].

There is ample evidence linking reduced but incomplete mitoticcheckpoint function with aneuploidy and tumourigenesis [Weaver B A andCleveland D W, Cancer Research, 2007, 67, 10103-5; King R W, Biochimicaet Biophysica Acta, 2008, 1786, 4-14]. In contrast, complete inhibitionof the mitotic checkpoint has been recognised to result in severechromosome missegregation and induction of apoptosis in tumour cells[Kops G J et al., Nature Reviews Cancer, 2005, 5, 773-85; Schmidt M andMedema R H, Cell Cycle, 2006, 5, 159-63; Schmidt M and Bastians H, DrugResistance Updates, 2007, 10, 162-81]. Therefore, mitotic checkpointabrogation through pharmacological inhibition of Mps-1 kinase or othercomponents of the mitotic checkpoint represents a new approach for thetreatment of proliferative disorders including solid tumours such ascarcinomas and sarcomas and leukaemias and lymphoid malignancies orother disorders associated with uncontrolled cellular proliferation.

Established anti-mitotic drugs such as vinca alkaloids, taxanes orepothilones activate the SAC inducing a mitotic arrest either bystabilising or destabilising microtubule dynamics. This arrest preventsseparation of sister chromatids to form the two daughter cells.Prolonged arrest in mitosis forces a cell either into mitotic exitwithout cytokinesis or into mitotic catastrophe leading to cell death.

In contrast, inhibitors of Mps-1 induce a SAC inactivation thataccelerates progression of cells through mitosis resulting in severechromosomal missegregation and finally in cell death.

These findings suggest that Mps-1 inhibitors should be of therapeuticvalue for the treatment of proliferative disorders associated withenhanced uncontrolled proliferative cellular processes such as, forexample, cancer, inflammation, arthritis, viral diseases,neurodegenerative diseases such as Alzheimer's disease, cardiovasculardiseases, or fungal diseases in a warm-blooded animal such as man.Therefore, inhibitors of Mps-1 represent valuable compounds that shouldcomplement therapeutic options either as single agents or in combinationwith other drugs.

Different compounds have been disclosed in prior art which show aninhibitory effect on Mps-1 kinase. WO2010/124826A1 discloses substitutedimidazoquinoxaline compounds as inhibitors of Mps-1 kinase.WO2011/026579A1 discloses substituted aminoquinoxalines as Mps-1inhibitors. WO2011/063908A1, WO2011/064328A1 as well as WO2011/063907 A1disclose triazolopyridine derivates as inhibitors of Mps-1 kinase.

Imidazopyridazine derivates have been disclosed for the treatment orprophylaxis of different diseases:

WO 2007/038314 A2 relates to fused heterocyclic compounds useful askinase modulators, including MK2 modulation. In particular, WO2007/038314 A2 relates to imidazo[1,2-b]pyridazines.

US patent application publication US 2008/0045536 A1 similarly relatesto fused heterocyclic compounds useful as kinase modulators, includingMK2 modulation. In particular, it relates to imidazo[1,2-b]pyridazines.

WO 2010/042699 A1 relates to fused heterocyclic compounds useful askinase modulators, particularly CK2 modulation. In particular, WO2010/042699 A1 relates to imidazo[1,2-b]pyridazines which aresubstituted with a nitrite group in position 3.

WO 2007/025090 A2 relates to heterocyclic compounds useful as inhibitorsof MEK kinase. In particular, WO 2007/025090 A2 relates inter alia toimidazo[1,2-b]pyridazines.

WO 1998/08847 A1 relates to heterocyclic compounds useful ascorticotropin releasing factor (hormone) CRF (CRH) antagonists. Inparticular, WO 1998/08847 A1 relates inter alia toimidazo[1,2-b]pyridazines.

WO 2011/013729A1 discloses fused imidazole derivatives as Mps-1inhibitors. Among the disclosed fused imidazole derivates there are alsoimidazo[1,2-b]pyridazines.

WO 2012/032031A1 inter alia is related to imidazo[1,2-b]pyridazines asMps-1 inhibitors.

However, the state of the art described above does not specificallydescribe the imidazopyridazine compounds of formula (A), (B) or (C), ora stereoisomer, a tautomer, an N-oxide, a hydrate, a solvate, or a saltthereof, or a mixture of same, as described and defined herein, and ashereinafter referred to as “compounds of the present invention”, ortheir pharmacological activity and stability.

It has now been found, and this constitutes the basis of the presentinvention, that said compounds of the present invention have surprisingand advantageous properties.

In particular, said compounds of the present invention have surprisinglybeen found to effectively inhibit Mps-1 kinase and may therefore be usedfor the treatment or prophylaxis of diseases of uncontrolled cellgrowth, proliferation and/or survival, inappropriate cellular immuneresponses, or inappropriate cellular inflammatory responses or diseaseswhich are accompanied with uncontrolled cell growth, proliferationand/or survival, inappropriate cellular immune responses, orinappropriate cellular inflammatory responses, particularly in which theuncontrolled cell growth, proliferation and/or survival, inappropriatecellular immune responses, or inappropriate cellular inflammatoryresponses is mediated by Mps-1 kinase, such as, for example,haemotological tumours, solid tumours, and/or metastases thereof, e.g.Leukaemias and myelodysplastic syndrome, malignant lymphomas, head andneck tumours including brain tumours and brain metastases, tumours ofthe thorax including non-small cell and small cell lung tumours,gastrointestinal tumours, endocrine tumours, mammary and othergynaecological tumours, urological tumours including renal, bladder andprostate tumours, skin tumours, and sarcomas, and/or metastases thereof.

The compounds of the present invention surprisingly exhibit a superioroverall profile with respect to Mps-1-related activity in a functionalassay (Spindle Assembly Checkpoint Assay), antiproliferative activity(Proliferation Assay with HeLa cells), metabolic stability (in vitrometabolic stability in rat hepatocytes) and drug-drug interactionpotential (inhibition of liver enzyme CYP3A4), as will be shownhereinafter.

SUMMARY OF THE INVENTION

The present invention covers compounds of formulae (A), (B), and (C):

or a stereoisomer, a tautomer, an N-oxide, a hydrate, a solvate, or asalt thereof, or a mixture of same.

The compounds of the present invention are characterized by: activity inSpindle Assembly Checkpoint Assay <1.0 nM, activity in ProliferationAssay with HeLa cells <25 nM, in vitro metabolic stability in rathepatocytes Fmax ≧39%, and inhibition of liver enzyme CYP3A4 ≧5 μM.

The present invention also relates to methods of preparing saidcompounds, to pharmaceutical compositions and combinations comprisingsaid compounds, to the use of said compounds for manufacturing apharmaceutical composition for the treatment or prophylaxis of adisease, as well as to intermediate compounds useful in the preparationof said compounds.

DETAILED DESCRIPTION OF THE INVENTION

The terms as mentioned in the present text have preferably the followingmeanings:

The term “halogen atom” or “halo-” is to be understood as meaning afluorine, chlorine, bromine or iodine atom.

The term “C₁-C₆-alkyl” is to be understood as preferably meaning alinear or branched, saturated, monovalent hydrocarbon group having 1, 2,3, 4, 5, or 6 carbon atoms, e.g. a methyl, ethyl, propyl, butyl, pentyl,hexyl, iso-propyl, iso-butyl, sec-butyl, tert-butyl, iso-pentyl,2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, 1,2-dimethylpropyl,neo-pentyl, 1,1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl,2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl,3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl,2,3-dimethylbutyl, 1,3-dimethylbutyl, or 1,2-dimethylbutyl group, or anisomer thereof. Particularly, said group has 1, 2, 3 or 4 carbon atoms(“C₁-C₄-alkyl”), e.g. a methyl, ethyl, propyl, butyl, iso-propyl,iso-butyl, sec-butyl, tert-butyl group, more particularly 1, 2 or 3carbon atoms (“C₁-C₃-alkyl”), e.g. a methyl, ethyl, n-propyl- oriso-propyl group.

The term “C₁-C₆”, as used throughout this text, e.g. in the context ofthe definition of “C₁-C₆-alkyl”, is to be understood as meaning an alkylgroup having a finite number of carbon atoms of 1 to 6, i.e. 1, 2, 3, 4,5, or 6 carbon atoms. It is to be understood further that said term“C₁-C₆” is to be interpreted as any sub-range comprised therein, e.g.C₁-C₆, C₂-C₅, C₃-C₄, C₁-C₂, C₁-C₃, C₁-C₄, C₁-C₅, C₁-C₆; particularlyC₁-C₂, C₁-C₃, C₁-C₄, C₁-C₅, C₁-C₆; more particularly C₁-C₄.

The term “substituted” means that one or more hydrogens on thedesignated atom is replaced with a selection from the indicated group,provided that the designated atom's normal valency under the existingcircumstances is not exceeded, and that the substitution results in astable compound. Combinations of substituents and/or variables arepermissible only if such combinations result in stable compounds.

The term “optionally substituted” means optional substitution with thespecified groups, radicals or moieties.

As used herein, the term “Leaving group” refers to an atom or a group ofatoms that is displaced in a chemical reaction as stable species takingwith it the bonding electrons. Preferably, a leaving group is selectedfrom the group comprising: halo, in particular chloro, bromo or iodo,methanesulfonyloxy, p-toluenesulfonyloxy, trifluoromethanesulfonyloxy,nonafluorobutanesulfonyloxy, (4-bromo-benzene)sulfonyloxy,(4-nitro-benzene)sulfonyloxy, (2-nitro-benzene)-sulfonyloxy,(4-isopropyl-benzene)sulfonyloxy,(2,4,6-tri-isopropyl-benzene)-sulfonyloxy,(2,4,6-trimethyl-benzene)sulfonyloxy, (4-tertbutyl-benzene)sulfonyloxy,benzenesulfonyloxy, and (4-methoxy-benzene)sulfonyloxy.

Where the plural form of the word compounds, salts, polymorphs,hydrates, solvates and the like, is used herein, this is taken to meanalso a single compound, salt, polymorph, isomer, hydrate, solvate or thelike.

By “stable compound” or “stable structure” is meant a compound that issufficiently robust to survive isolation to a useful degree of purityfrom a reaction mixture, and formulation into an efficacious therapeuticagent.

In accordance with a first aspect, the present invention is directed toa compound of formula (A), (B) or (C):

In a preferred embodiment, the invention relates to a compound offormula (A), supra.

In another preferred embodiment, the invention relates to a compound offormula (B), supra.

In another preferred embodiment, the invention relates to a compound offormula (C), supra.

In an embodiment of the above-mentioned aspect, the invention relates toa compound of formulae (A), (B) or (C) in the form of a tautomer, anN-oxide, a hydrate, a solvate, or a salt thereof, or a mixture of same.

The present invention includes all possible stereoisomers of thecompounds of the present invention as single stereoisomers, or as anymixture of said stereoisomers, in any ratio. Isolation of a singlestereoisomer, e.g. a single enantiomer or a single diastereomer, of acompound of the present invention may be achieved by any suitable stateof the art method, such as chromatography, especially chiralchromatography, for example.

Further, the compounds of the present invention may exist as tautomers.For example, any compound of the present invention which contains apyrazole moiety as a heteroaryl group for example can exist as a 1Htautomer, or a 2H tautomer, or even a mixture in any amount of the twotautomers, or a triazole moiety for example can exist as a 1H tautomer,a 2H tautomer, or a 4H tautomer, or even a mixture in any amount of said1H, 2H and 4H tautomers, namely:

The present invention includes all possible tautomers of the compoundsof the present invention as single tautomers, or as any mixture of saidtautomers, in any ratio.

The invention also includes all suitable isotopic variations of acompound of the invention. An isotopic variation of a compound of theinvention is defined as one in which at least one atom is replaced by anatom having the same atomic number but an atomic mass different from theatomic mass usually or predominantly found in nature. Examples ofisotopes that can be incorporated into a compound of the inventioninclude isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus,sulphur, fluorine, chlorine, bromine and iodine, such as ²H (deuterium),³H (tritium), ¹¹C ¹³C, ¹⁴C, ¹⁵N, ¹⁷O, ¹⁸O, ³²P, ³³P, ³³S, ³⁴S, ³⁵S, ³⁶S,¹⁸F, ³⁶Cl, ⁸²Br, ¹²³I, ¹²⁴I, ¹²⁹I and ¹³¹I, respectively. Certainisotopic variations of a compound of the invention, for example, thosein which one or more radioactive isotopes such as ³H or ¹⁴C areincorporated, are useful in drug and/or substrate tissue distributionstudies. Tritiated and carbon-14, i.e., ¹⁴C, isotopes are particularlypreferred for their ease of preparation and detectability. Further,substitution with isotopes such as deuterium may afford certaintherapeutic advantages resulting from greater metabolic stability, forexample, increased in vivo half-life or reduced dosage requirements andhence may be preferred in some circumstances. Isotopic variations of acompound of the invention can generally be prepared by conventionalprocedures known by a person skilled in the art such as by theillustrative methods or by the preparations described in the exampleshereafter using appropriate isotopic variations of suitable reagents.

Further, the compounds of the present invention can exist as N-oxides,which are defined in that at least one nitrogen of the compounds of thepresent invention is oxidised. The present invention includes all suchpossible N-oxides.

Furthermore, the present invention includes all possible crystallineforms, or polymorphs, of the compounds of the present invention, eitheras single polymorphs, or as a mixture of more than one polymorph, in anyratio.

The compounds of the present invention can exist as a hydrate, or as asolvate, wherein the compounds of the present invention contain polarsolvents, in particular water, methanol or ethanol for example asstructural element of the crystal lattice of the compounds. The amountof polar solvents, in particular water, may exist in a stoichiometric ornon-stoichiometric ratio. In the case of stoichiometric solvates, e.g. ahydrate, hemi-, (semi-), mono-, sesqui-, di-, tri-, tetra-, penta- etc.solvates or hydrates, respectively, are possible. The present inventionincludes all such hydrates or solvates.

The present invention also relates to useful forms of the compounds asdisclosed herein, such as metabolites, hydrates, solvates, prodrugs,salts, in particular pharmaceutically acceptable salts, in vivohydrolysable esters, and co-precipitates. The term “pharmaceuticallyacceptable salt” refers to a relatively non-toxic, inorganic or organicacid addition salt of a compound of the present invention. For example,see S. M. Berge, et al. “Pharmaceutical Salts,” J. Pharm. Sci. 1977, 66,1-19.

A suitable pharmaceutically acceptable salt of the compounds of thepresent invention may be, for example, an acid-addition salt of acompound of the present invention bearing a nitrogen atom, in a chain orin a ring, for example, which is sufficiently basic, such as anacid-addition salt with an inorganic acid, such as hydrochloric,hydrobromic, hydroiodic, sulfuric, bisulfuric, phosphoric, or nitricacid, for example, or with an organic acid, such as formic, acetic,acetoacetic, pyruvic, trifluoroacetic, propionic, butyric, hexanoic,heptanoic, undecanoic, Lauric, benzoic, salicylic,2-(4-hydroxybenzoyl)-benzoic, camphoric, cinnamic,cyclopentanepropionic, digluconic, 3-hydroxy-2-naphthoic, nicotinic,pamoic, pectinic, persulfuric, 3-phenylpropionic, picric, pivalic,2-hydroxyethanesulfonate, itaconic, sulfamic, trifluoromethanesulfonic,dodecylsulfuric, ethansulfonic, benzenesulfonic, para-toluenesulfonic,methansulfonic, 2-naphthalenesulfonic, naphthalinedisulfonic,camphorsulfonic acid, citric, tartaric, stearic, lactic, oxalic,malonic, succinic, malic, adipic, alginic, maleic, fumaric, D-gluconic,mandelic, ascorbic, glucoheptanoic, glycerophosphoric, aspartic,sulfosalicylic, hemisulfuric, or thiocyanic acid, for example.

Further, another suitably pharmaceutically acceptable salt of a compoundof the present invention which is sufficiently acidic, is an alkalimetal salt, for example a sodium or potassium salt, an alkaline earthmetal salt, for example a calcium or magnesium salt, an ammonium salt ora salt with an organic base which affords a physiologically acceptablecation, for example a salt with N-methyl-glucamine, dimethyl-glucamine,ethyl-glucamine, Lysine, dicyclohexylamine, 1,6-hexadiamine,ethanolamine, glucosamine, sarcosine, serinol,tris-hydroxy-methyl-aminomethane, aminopropandiol, sovak-base,1-amino-2,3,4-butantriol. Additionally, basic nitrogen containing groupsmay be quaternised with such agents as lower alkyl halides such asmethyl, ethyl, propyl, and butyl chlorides, bromides and iodides;dialkyl sulfates like dimethyl, diethyl, and dibutyl sulfate; and diamylsulfates, long chain halides such as decyl, lauryl, myristyl andstrearyl chlorides, bromides and iodides, aralkyl halides like benzyland phenethyl bromides and others.

Those skilled in the art will further recognise that acid addition saltsof the claimed compounds may be prepared by reaction of the compoundswith the appropriate inorganic or organic acid via any of a number ofknown methods. Alternatively, alkali and alkaline earth metal salts ofacidic compounds of the invention are prepared by reacting the compoundsof the invention with the appropriate base via a variety of knownmethods.

The present invention includes all possible salts of the compounds ofthe present invention as single salts, or as any mixture of said salts,in any ratio.

As used herein, the term “in vivo hydrolysable ester” is understood asmeaning an in vivo hydrolysable ester of a compound of the presentinvention containing a carboxy or hydroxy group, for example, apharmaceutically acceptable ester which is hydrolysed in the human oranimal body to produce the parent acid or alcohol. Suitablepharmaceutically acceptable esters for carboxy include for examplealkyl, cycloalkyl and optionally substituted phenylalkyl, in particularbenzyl esters, C₁-C₆ alkoxymethyl esters, e.g. methoxymethyl, C₁-C₆alkanoyloxymethyl esters, e.g. pivaloyloxymethyl, phthalidyl esters,C₃-C₈ cycloalkoxy-carbonyloxy-C₁-C₆ alkyl esters, e.g.1-cyclohexylcarbonyloxyethyl; 1,3-dioxolen-2-onylmethyl esters, e.g.5-methyl-1,3-dioxolen-2-onylmethyl; and C₁-C₆-alkoxycarbonyloxyethylesters, e.g. 1-methoxycarbonyloxyethyl, and may be formed at any carboxygroup in the compounds of this invention.

An in vivo hydrolysable ester of a compound of the present inventioncontaining a hydroxy group includes inorganic esters such as phosphateesters and [alpha]-acyloxyalkyl ethers and related compounds which as aresult of the in vivo hydrolysis of the ester breakdown to give theparent hydroxy group. Examples of [alpha]-acyloxyalkyl ethers includeacetoxymethoxy and 2,2-dimethylpropionyloxymethoxy. A selection of invivo hydrolysable ester forming groups for hydroxy include alkanoyl,benzoyl, phenylacetyl and substituted benzoyl and phenylacetyl,alkoxycarbonyl (to give alkyl carbonate esters), dialkylcarbamoyl andN-(dialkylaminoethyl)-N-alkylcarbamoyl (to give carbamates),dialkylaminoacetyl and carboxyacetyl. The present invention covers allsuch esters.

The compounds of the present invention have surprisingly been found toeffectively inhibit Mps-1 kinase and may therefore be used for thetreatment or prophylaxis of diseases of uncontrolled cell growth,proliferation and/or survival, inappropriate cellular immune responses,or inappropriate cellular inflammatory responses or diseases which areaccompanied with uncontrolled cell growth, proliferation and/orsurvival, inappropriate cellular immune responses, or inappropriatecellular inflammatory responses, particularly in which the uncontrolledcell growth, proliferation and/or survival, inappropriate cellularimmune responses, or inappropriate cellular inflammatory responses ismediated by Mps-1 kinase, such as, for example, haemotological tumours,solid tumours, and/or metastases thereof, e.g. Leukaemias andmyelodysplastic syndrome, malignant lymphomas, head and neck tumoursincluding brain tumours and brain metastases, tumours of the thoraxincluding non-small cell and small cell lung tumours, gastrointestinaltumours, endocrine tumours, mammary and other gynaecological tumours,urological tumours including renal, bladder and prostate tumours, skintumours, and sarcomas, and/or metastases thereof.

Therefore, the compounds of formulae (A), (B), and (C), supra, areexpected to be valuable as therapeutic agents.

Accordingly, in another embodiment, the present invention is directed toa compound of formula (A), (B) or (C), supra, or a stereoisomer, atautomer, an N-oxide, a hydrate, a solvate, or a salt thereof,particularly a pharmaceutically acceptable salt thereof, or a mixture ofsame, for use in the treatment or prophylaxis of a disease.

In another embodiment, the present invention provides a method oftreating disorders associated with enhanced uncontrolled proliferativecellular processes in a patient in need of such treatment, comprisingadministering to the patient an effective amount of a compound offormula (A), (B) or (C), supra.

The term “treating” or “treatment” as stated throughout this document isused conventionally, e.g., the management or care of a subject for thepurpose of combating, alleviating, reducing, relieving, improving thecondition of a disease or disorder, such as a carcinoma.

The term “subject” or “patient” includes organisms which are capable ofsuffering from a cell proliferative disorder or who could otherwisebenefit from the administration of a compound of the invention, such ashuman and non-human animals. Preferred humans include human patientssuffering from or prone to suffering from a cell proliferative disorderor associated state, as described herein. The term “non-human animals”includes vertebrates, e.g., mammals, such as non-human primates, sheep,cow, dog, cat and rodents, e.g., mice, and non-mammals, such aschickens, amphibians, reptiles, etc.

The terms “cell proliferative disorder” or “disorder associated withenhanced uncontrolled proliferative cellular processes” includedisorders involving the undesired or uncontrolled proliferation of acell. The compounds of the present invention can be utilized to prevent,inhibit, block, reduce, decrease, control, etc., cell proliferationand/or cell division, and/or produce apoptosis. This method comprisesadministering to a subject in need thereof, including a mammal,including a human, an amount of a compound of this invention, or apharmaceutically acceptable salt, isomer, polymorph, metabolite, hydrateor solvate thereof which is effective to treat or prevent the disorder.

In another embodiment, the present invention is directed to a compoundof formula (A), (B) or (C), or a stereoisomer, a tautomer, an N-oxide, ahydrate, a solvate, or a salt thereof, particularly a pharmaceuticallyacceptable salt thereof, or a mixture of same, for use in the treatmentor prophylaxis of a disease, wherein said disease is a disease ofuncontrolled cell growth, proliferation and/or survival, aninappropriate cellular immune response, or an inappropriate cellularinflammatory response, particularly in which the uncontrolled cellgrowth, proliferation and/or survival, inappropriate cellular immuneresponse, or inappropriate cellular inflammatory response is mediated bythe mitogen-activated protein kinase (MEK-ERK) pathway, moreparticularly in which the disease of uncontrolled cell growth,proliferation and/or survival, inappropriate cellular immune response,or inappropriate cellular inflammatory response is a haemotologicaltumour, a solid tumour and/or metastases thereof, e.g. Leukaemias andmyelodysplastic syndrome, malignant lymphomas, head and neck tumoursincluding brain tumours and brain metastases, tumours of the thoraxincluding non-small cell and small cell lung tumours, gastrointestinaltumours, endocrine tumours, mammary and other gynaecological tumours,urological tumours including renal, bladder and prostate tumours, skintumours, and sarcomas, and/or metastases thereof.

Compounds of formulae (A), (B), and (C), supra, may be administered asthe sole pharmaceutical agent or in combination with one or moreadditional therapeutic agents where the combination causes nounacceptable adverse effects. This combination therapy includesadministration of a single pharmaceutical dosage formulation whichcontains a compound of formula (A), (B) or (C) and one or moreadditional therapeutic agents, as well as administration of the compoundof formula (A), (B) or (C) and each additional therapeutic agent in itsown separate pharmaceutical dosage formulation. For example, a compoundof formula (A), (B) or (C) and a therapeutic agent may be administeredto the patient together in a single oral dosage composition such as atablet or capsule, or each agent may be administered in separate dosageformulations.

Where separate dosage formulations are used, the compound of formula(A), (B) or (C) and one or more additional therapeutic agents may beadministered at essentially the same time (e.g., concurrently) or atseparately staggered times (e.g., sequentially).

In another aspect, the invention provides a pharmaceutical compositioncomprising a compound of general formula (A), (B) or (C), or astereoisomer, a tautomer, an N-oxide, a hydrate, a solvate, or a saltthereof, particularly a pharmaceutically acceptable salt thereof, or amixture of same, and a pharmaceutically acceptable diluent or carrier.

Preferably, the pharmaceutical combination comprises:

-   -   one or more compounds of general formula (A), (B) or (C), or a        stereoisomer, a tautomer, an N-oxide, a hydrate, a solvate, or a        salt thereof, particularly a pharmaceutically acceptable salt        thereof, or a mixture of same; and    -   one or more agents selected from: a taxane, such as Docetaxel,        Paclitaxel, or Taxol; an epothilone, such as Ixabepilone,        Patupilone, or Sagopilone; Mitoxantrone; Predinisolone;        Dexamethasone; Estramustin; Vinblastin; Vincristin; Doxorubicin;        Adriamycin; Idarubicin; Daunorubicin; Bleomycin; Etoposide;        Cyclophosphamide; Ifosfamide; Procarbazine; Melphalan;        5-Fluorouracil; Capecitabine; Fludarabine; Cytarabine; Ara-C;        2-Chloro-2-deoxyadenosine; Thioguanine; an anti-androgen, such        as Flutamide, Cyproterone acetate, or Bicalutamide; Bortezomib;        a platinum derivative, such as Cisplatin, or Carboplatin;        Chlorambucil; Methotrexate; and Rituximab.

In still another aspect, the invention provides a process for preparinga pharmaceutical composition. The process includes the step of combiningat least one compound of formula (A), (B) or (C) as defined above withat least one pharmaceutically acceptable carrier, and bringing theresulting combination into a suitable administration form.

In still another aspect, the invention provides use of a compound offormula (A), (B) or (C) as defined above for manufacturing apharmaceutical composition for the treatment or prevention of a cellproliferative disorder. In certain embodiments, the cell proliferativedisorder is cancer.

The active component of formula (A), (B) or (C) can act systemicallyand/or locally. For this purpose, it can be applied in a suitablemanner, for example orally, parenterally, pulmonally, nasally,sublingually, lingually, buccally, rectally, transdermally,conjunctivally, optically, or as an implant or stent.

When the compounds of the present invention are administered aspharmaceuticals, to humans and animals, they can be given per se or as apharmaceutical composition containing, for example, 0.1 to 99.5% (morepreferably, 0.5 to 90%) of active ingredient in combination with apharmaceutically acceptable carrier.

Regardless of the route of administration selected, the compounds of theinvention, which may be used in a suitable hydrated form, and/or thepharmaceutical compositions of the present invention, are formulatedinto pharmaceutically acceptable dosage forms by conventional methodsknown to those of skill in the art.

Actual dosage levels and time course of administration of the activeingredients in the pharmaceutical compositions of the invention may bevaried so as to obtain an amount of the active ingredient which iseffective to achieve the desired therapeutic response for a particularpatient, composition, and mode of administration, without being toxic tothe patient.

In accordance with another aspect, the present invention covers methodsof preparing compounds of the present invention.

In accordance with a first embodiment, the present invention alsorelates to a method of preparing a compound of formula (A), (B) or (C),supra, said method comprising the step of allowing an intermediatecompound of general formula (A1), (B1) or (C1):

in which Z′ represents a group selected from: —C(═O)OH and—C(═O)O—(C₁-C₆-alkyl); to react with a compound of formula Ib:

thereby giving, upon optional deprotection, a compound of formula (A),(B) or (C).

In accordance with another embodiment, the present invention alsorelates to a method of preparing a compound of formula (A), (B) or (C),supra, said method comprising the step of allowing an intermediatecompound of formula (A2), (B2) or (C2):

in which Q² is a leaving group, preferably Q² is a halogen atom;to react with a compound of general formula IIa:

in which Y is a substituent which is displaced in a coupling reaction,such as a boronic acid group, or an ester of a boronic acid group, forexample; thereby giving, upon optional deprotection, a compound offormula (A), (B) or (C).

In accordance with another embodiment, the present invention alsorelates to a method of preparing a compound of formula (A), (B) or (C),supra, said method comprising the step of allowing an intermediatecompound of formula (A3), (B3) or (C3):

in which Q¹ is a leaving group, for example a halogen atom;to react with a compound of general formula (A4), (B4) or (C4):

thereby giving, upon optional deprotection, a compound of generalformula (A), (B) or (C):

In accordance with another embodiment, the present invention alsorelates to a method of preparing a compound of formula (A), (B) or (C),supra, said method comprising the step of allowing an intermediatecompound of formula (A5), (B5) or (C5):

in which Q³ is an optionally protected NH₂-group;to react with a compound of general formula (A6), (B6) or (C6):

thereby giving, after reduction of the imine, and upon optionaldeprotection, a compound of formula (A), (B) or (C):

In accordance with another embodiment, the present invention relates toa method of preparing a compound of formula (A), (B) or (C), said methodcomprising the step of allowing an intermediate compound of formula(A7), (B7) or (C7):

in which R^(3′) is a leaving group;to react with a compound of formula (A8), (B8) or (C8):

in which Y is a substituent which is displaced in a coupling reaction,such as a hydrogen atom, or a boronic acid group, or a boronic estergroup, for example;thereby giving a compound of formula (A), (B) or (C):

In accordance with a further aspect, the present invention coversintermediate compounds which are useful in the preparation of compoundsof the present invention of formula (A), (B) or (C), particularly in themethod described herein.

In particular, the present invention covers intermediate compounds offormula (A1), (B1) and (C1):

in which Z′ represents a group selected from: —C(═O)OH and—C(═O)O—(C₁-C₆-alkyl).

In accordance with yet another aspect, the present invention coversintermediate compounds of formula (A2), (B2) or (C2):

in Q² is a leaving group, preferably Q² is a halogen atom.

In accordance with a further aspect, the present invention coversintermediate compounds of formula (A3), (B3) or (C3):

in which Q¹ is a leaving group, for example a halogen atom.

In accordance with a further aspect, the present invention coversintermediate compounds of formula (A5), (B5) or (C5):

in which Q³ is an optionally protected NH₂-group.

In accordance with a further aspect, the present invention coversintermediate compounds of general formula (A7), (B7) or (C7):

in which R^(3′) is a leaving group.

In accordance with a further aspect, the present invention relates tothe use of a compound of formula (A1), (A2), (A3), (A4), (A5), (A6),(A7) or (A8), supra, for the preparation of a compound of formula (A),supra.

In accordance with a further aspect, the present invention relates tothe use of a compound of formula (B1), (B2), (B3), (B4), (B5), (B6),(B7) or (B8), supra, for the preparation of a compound of formula (B),supra.

In accordance with a further aspect, the present invention relates tothe use of a compound of formula (C1), (C2), (C3), (C4), (C5), (C6),(C7) or (C8), supra, for the preparation of a compound of formula (C),supra.

EXPERIMENTAL SECTION General

The following Table lists the abbreviations used in this paragraph, andin the Examples section.

Abbreviation Meaning EDC 1-Ethyl-3-(3-dimethyllaminopropyl)carbodiimideDIPEA N,N-diisopropylethylamine DMF N,N-Dimethylformamide DMSO Dimethylsulfoxide DIPEA Diisopropylethylamine Pd(dppf)Cl₂Dichloro[1,1′-bis(diphenylphosphino)ferrocene]palladi- um(II) NMRNuclear magnetic resonance spectroscopy rt Room temperature RT Retentiontime in minutes MW Molecular weight NMP N-methylpyrrolidinone OxonePotassium peroxymonosulfate UPLC Ultra performance liquid chromatographySynthesis of the Compounds of the Present Invention

Compounds of formula (A), (B) or (C) can be synthesized as depicted inthe Scheme, in which

A represents

R³ represents

R^(3′) represents a leaving group;R⁵ represents

Q¹ represents a leaving group or an optionally protected NH₂-group; andQ² represents a leaving group;wherein * indicates the point of attachment of said groups with the restof the molecule.

Examples for typical leaving groups include but are not limited tohalogen atoms like a chlorine, bromine or iodine atom, or a groupselected from: methanesulfonyloxy, p-toluenesulfonyloxy,trifluoromethanesulfonyloxy, nonafluorobutanesulfonyloxy,(4-bromo-benzene)sulfonyloxy, (4-nitro-benzene)sulfonyloxy,(2-nitro-benzene)-sulfonyloxy, (4-isopropyl-benzene)sulfonyloxy,(2,4,6-tri-isopropyl-benzene)-sulfonyloxy,(2,4,6-trimethyl-benzene)sulfonyloxy, (4-tertbutyl-benzene)sulfonyloxy,benzenesulfonyloxy, and (4-methoxy-benzene)sulfonyloxy.

The Scheme exemplifies routes that allow variations for R³, R^(3′), R⁵,Q¹, Q² and A during the synthesis. Functional moieties in R³, R^(3′),R⁵, Q¹, Q² and A can be converted at every suitable stage of thesynthesis.

Compounds of formula X may be commercially available or can besynthesized according to procedures known to persons skilled in the art,for example applying procedures described in WO2007/38314A2.

A leaving group Q² can be introduced in compounds of general formula X,VI or III by procedures known to persons skilled in the art to givecompounds of general formula IX, V or II. As an example, halogens can beintroduced using halogenation reagents like N-iodosuccinimide (NIS),N-bromosuccinimide (NBS), or N-chlorosuccinimide (NCS), in an inertsolvent like N,N-dimethylformamide or 1-methylpyrrolidin-2-one, forexample, at temperatures ranging from room temperature to the boilingpoint of the solvent, for example.

Compounds of general formula I, IV or VIII can be obtained fromcompounds of general formula II, V or IX via a coupling reaction betweena reagent of formula Y-A, in which A is defined supra and Y represents asuitable functional group by which the group A can be transferred to theQ-group bearing carbon atom of compounds of formula II, V or IX.Examples of suitable functional groups for Y in A-Y include boronicacids A-B(OH)₂, or esters of boronic acids A-B(OC₁-C₆-alkyl)₂. Saidcoupling reactions are performed in the presence of suitable catalysts,such as, for example, palladium based catalysts like, for example,Palladium (II) acetate, tetrakis(triphenylphosphine)palladium (0),bis(triphenylphosphine)-palladium (II) chloride or(1,1,-bis(diphenylphosphino) ferrocene)-dichloropalladium (II) andoptionally suitable additives such as, for example, phosphines like, forexample, P(oTol)₃ or triphenylphosphine and optionally with a suitablebase, such as, for example, potassium carbonate, sodium2-methylpropan-2-olate, tetrabutylammonium fluoride or tribasicpotassium phosphate in a suitable solvent, such as, for example,tetrahydrofuran.

Examples of such coupling reactions may be found in the textbookentitled “Metal-Catalyzed Cross-Coupling Reactions”, Armin de Meijere(Editor), François Diederich (Editor) September 2004, Wiley InterscienceISBN: 978-3-527-30518-6.

Compounds of general formula I, II, III or VII can be obtained fromcompounds of general formula IV, V, VI or VIII via a coupling reactionusing a reagent of formula Y—R³ in which R³ is defined supra and Yrepresents a suitable functional group by which the group R³ can betransferred to the R^(3′) bearing carbon atom of compounds of formulaIV, V, VI or VIII. Examples of suitable functional groups Y for the usein coupling reactions are given supra for the preparation of compoundsof general formula I, IV or VIII from compounds of general formula II, Vor IX.

Y in Y—R³ may represent an acidic hydrogen that can be removed bysuitable bases, for example sodium hydride, in a suitable solvent, suchas DMSO or tetrahydrofuran at temperatures ranging from rt to theboiling point. The resulting nucleophiles, phenolates, can be used toreplace R^(3′) in compounds of general formula IV, V, VI or VIII to addphenol ethers to give compounds of general formula I, II, III or VII.

Compounds of general formula I, II, III or VII containing phenoletherscan also be built by Ullmann-type coupling reactions in the presence ofsuitable catalysts, such as, for example, copper based catalysts likecopper(II)diacetate in presence of a suitable base, like for example,caesium carbonate staring from compounds of general formula IV, V, VI orVIII in which R^(3′) represents a leaving group such as, for example, aniodine, bromine or chlorine atom. Optionally, suitable ligands likeN,N-dimethylglycine or phenyl hydrogen pyrrolidin-2-ylphosphonate can beadded.

In the case Q¹ represents a leaving group, the introduction of aR⁵—CH₂-group can be achieved by nucleophilic substitution of Q¹ incompounds of formula VII, VIII, IX or X i.e. by a reaction with suitableamines R⁵—CH₂—NH₂ in the presence of a suitable base, such as, forexample DIPEA in a suitable solvent such as N,N-dimethylformamide or1-methylpyrrolidin-2-one, at temperatures ranging from room temperatureto the boiling point of the solvent to give amines of general formula I,IV, V or VI.

In the case Q¹ represents a leaving group, the introduction of aR⁵—CH₂-group can also be achieved in a coupling reaction in which Q¹ incompounds of formula VII, VIII, IX or X is reacted with suitable aminesR⁵—CH₂—NH₂ optionally in the presence of a suitable catalyst, such asPd₂dba₃ and BINAP for example, and optionally with a suitable base, suchas, for example, sodium tert-butylate in a suitable solvent, such as,for example, N,N-dimethylformamide or 1-methylpyrrolidin-2-one to giveamines of general formula I, IV, V or VI.

In the case Q¹ represents an optionally protected NH₂-group theintroduction of a R⁵—CH₂-group, after deprotection to a NH₂-group, canbe achieved by a reductive amination reaction using an aldehyde offormula O═CHR⁵, a suitable reducing agent, for example sodiumtris(acetato-kappaO)(hydrido)borate or sodium cyanoborohydride in asuitable solvent like, for example, acetic acid at reaction temperaturesranging from room temperature to the boiling point of the solvent.Residues in compounds of formula I, II, III, IV, V, VI, VII, VIII, IX orX can be optionally modified. For example, thioethers can be oxidizedusing oxidation reagents like 3-chlorobenzenecarboperoxoic acid, oxoneor dimethyldioxirane in inert solvents like dichloromethane or acetone,respectively. Depending on the stoichiometric ratio of oxidation reagentto the afore mentioned compounds sulfoxides or sulfones or mixturesthereof will be obtained.

The compounds and intermediates produced according to the methods of theinvention may require purification. Purification of organic compounds iswell known to the person skilled in the art and there may be severalways of purifying the same compound. In some cases, no purification maybe necessary. In some cases, the compounds may be purified bycrystallisation. In some cases, impurities may be removed by stirringusing a suitable solvent. In some cases, the compounds may be purifiedby chromatography, particularly flash chromatography, using for examplepre-packed silica gel cartridges, e.g. from Separtis such as Isolute®Flash silica gel or Isolute® Flash NH2 silica gel in combination with asuitable chromatographic system such as a Flashmaster II (Separtis) oran Isolera system (Biotage) and eluents such as, for example, gradientsof hexane/EtOAc or DCM/methanol. In some cases, the compounds may bepurified by preparative HPLC using, for example, a Waters autopurifierequipped with a diode array detector and/or on-line electrosprayionisation mass spectrometer in combination with a suitable pre-packedreverse phase column and eluants such as, for example, gradients ofwater and acetonitrile which may contain additives such astrifluoroacetic acid, formic acid or aqueous ammonia.

Example 1N-cyclopropyl-4-{6-(3-fluoro-4-methoxyphenoxy)-8-[(oxetan-3-ylmethyl)amino]imidazo[1,2-b]pyridazin-3-yl}-2-methylbenzamide

A solution of 128 mg (900 μmol) of 3-fluoro-4-methoxyphenol in 2 mL ofdimethylsulfoxide was treated with 36 mg (900 μmol) of sodium hydrideand stirred at room temperature for 1 hour. Then 68 mg (150 μmol) of4-{6-bromo-8-[(oxetan-3-ylmethyl)amino]imidazo[1,2-b]pyridazin-3-yl}-N-cyclopropyl-2-methylbenzamide,which was prepared according to intermediate example 1a, was added andthe mixture was heated for 1 h at 130° C. and overnight at 120° C. togive after HPLC purification 17 mg (20%) of the title compound.

¹H-NMR (DMSO-d6): δ=0.43-0.50 (2H), 0.59-0.68 (2H), 2.11 (3H), 2.77(1H), 3.35 (1H), 3.61 (2H), 3.83 (3H), 4.34 (2H), 4.63 (2H), 6.09 (1H),7.02-7.09 (1H), 7.13-7.25 (2H), 7.30 (1H), 7.61-7.68 (1H), 7.77 (1H),7.83 (1H), 7.91 (1H), 8.22 (1H) ppm.

Intermediate Example 1a4-{6-bromo-8-[(oxetan-3-ylmethyl)amino]imidazo[1,2-b]pyridazin-3-yl}-N-cyclopropyl-2-methylbenzamide

To a suspension of 3000 mg (6677 μmol)4-[6-bromo-8-(methylsulfonyl)imidazo[1,2-b]pyridazin-3-yl]-N-cyclopropyl-2-methylbenzamide,which was prepared according to intermediate example 1b in 150 mL of THFwere added 873 mg (1002 μmol) 1-(oxetan-3-yl)methanamine and 2589 mg(2003 μmol) DIPEA and the mixture was heated for 72 hours at 60° C.After further addition of 100 mg 1-(oxetan-3-yl)methanamine and heatingfor 8 h at 60° C., the solvent was removed in vaccuo and the residue wastaken up in ethyl acetate and washed with water. The precipitate formedwas filtered off to yield 0.99 g (33%) of the title compound. Theremaining aqueous phase was reextracted with DCM, and the combinedorganic phases were evaporated. The residue was triturated with THF at75° C. to yield another 1.65 g (53%) of the title compound.

¹H-NMR (DMSO-d6): δ=0.46-0.53 (2H), 0.61-0.69 (2H), 2.35 (3H), 2.75-2.85(1H), 3.25 (1H), 3.54-3.69 (2H), 4.31 (2H), 4.62 (2H), 6.45 (1H), 7.36(1H), 7.84 (1H), 7.90 (1H), 7.94 (1H), 8.12 (1H), 8.30 (1H) ppm.

Intermediate Example 1b4-[6-bromo-8-(methylsulfonyl)imidazo[1,2-b]pyridazin-3-yl]-N-cyclopropyl-2-methylbenzamide

To a solution of 12.5 g (28.75 mmol)4-[6-bromo-8-(methylsulfanyl)imidazo[1,2-b]pyridazin-3-yl]-N-cyclopropyl-2-methylbenzamide,which was prepared according to intermediate example 1c in 400 mL of DMFwere added 53.03 g (86.26 mmol) potassium hydrogen sulfate sulfate(hydroperoxysulfonyl)oxidanide (5:1:1:2) and the mixture was stirredovernight at rt. to give, after aqueous work-up, 8.6 g (60%) of thetitle compound (impurity is4-[6-bromo-8-(methylsulfinyl)imidazo[1,2-b]pyridazin-3-yl]-N-cyclopropyl-2-methylbenzamide)UPLC-MS: RT=0.98 min; m/z (ES+) 450.3 [MH+]; required MW=449.3.

Intermediate Example 1c4-[6-bromo-8-(methylsulfanyl)imidazo[1,2-b]pyridazin-3-yl]-N-cyclopropyl-2-methylbenzamide

A mixture comprising 55.69 g (150 mmol)6-bromo-3-iodo-8-(methylsulfanyl)imidazo[1,2-b]pyridazine which wasprepared according to intermediate example 1d, 68 g (225 mmol)N-cyclopropyl-2-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzamide,which was prepared according to intermediate example 1 g, 11 g (15 mmol)(1,1,-bis(diphenylphosphino)ferrocene)-dichloropalladium (11), 450 mLaqueous 1M potassium carbonate solution and 632 mL tetrahydrofuran wasstirred at 60° C. for 12 hours to give, after aqueous workup, 130 gcrude product. The residue was triturated with DCM to yield 15.15 g(24%) of the title compound. The filtrate was purified by chromatographyto give another 2.65 g (3%) of the title compound.

¹H-NMR (DMSO-d6): δ=0.47-0.54 (2H), 0.62-0.71 (2H), 2.36 (3H), 2.63(3H), 2.77-2.85 (1H), 7.17 (1H), 7.40 (1H), 7.85 (1H), 7.90 (1H), 8.12(1H), 8.31 (1H) ppm.

Intermediate Example 1d6-bromo-3-iodo-8-(methylsulfanyl)imidazo[1,2-b]pyridazine

To a solution of 174 g (432 mmol)6,8-dibromo-3-iodoimidazo[1,2-b]pyridazine which was prepared accordingto intermediate example 1e in 3.8 L dioxane were added 30.28 g (432mmol) sodium methanethiolate and the mixture was stirred at 60° C. for 5days. Further 25 g sodium methanethiolate were added and the mixture wasstirred at 80° C. for 2 h. After cooling, the solution was poured on 4 Lwater Water and the aqueous phase was extracted with ethyl acetate. Theorganic phase was washed with water, dried over sodium sulphate,filtered and evaporated to give 102 g (64%) of the title compound.

¹H-NMR (DMSO-d6): δ=6.79 (1H), 7.67 (1H) ppm.

Intermediate Example 1e 6,8-dibromo-3-iodoimidazo[1,2-b]pyridazine

To a mixture comprising 156 g (563 mmol)6,8-dibromoimidazo[1,2-b]pyridazine which was prepared according tointermediate example 1f, 190 g (1637 mmol) N-iodosuccinimide and 1.3 Lchloroform were added 5.5 mL HCl conc and the suspension was heated at70° C. overnight. The precipitate was filtered off and triturated withdiisopropylether to give 119 g (52%) of the title compound.

¹H-NMR (DMSO-d6): δ=7.92 (1H), 8.00 (1H) ppm.

Intermediate Example 1f 6,8-dibromoimidazo[1,2-b]pyridazine

A mixture comprising 235 g (931 mmol) 4,6-dibromopyridazin-3-amine whichwas prepared according to intermediate example 1 g, 421 mL (2792 mmol)2-bromo-1,1-diethoxyethane, 2.93 L water and 227 mL THF was heated at125° C. for h and at rt overnight. The solution was neutralized byaddition of solid NaHCO₃, the precipitate was filtered off, washed withwater and dried to give 156 g (80%) of the title compound as a brownishsolid.

¹H-NMR (DMSO-d6): δ=7.81 (1H), 8.40 (1H) ppm.

Intermediate Example 1 g 6,8-dibromoimidazo[1,2-b]pyridazine

To a mixture comprising 285 g (1638 mmol) 6-bromopyridazin-3-amine whichwas prepared according to intermediate example 1 h, 275 g (3276 mmol)NaHCO₃ and 2815 mL MeOH was dropwise added 85 mL (1638 mmol) bromine atrt and it was stirred at rt overnight. After further addition of 34 mL(655 mmol) bromine and 55 g (655 mmol) NaHCO₃, the mixture was stirredovernight again. The solvent was reduced to about 1000 mL and themixture was poured on 5 L of water. The precipitate was filtered off,washed with water and dried give 411 g (99%) of the title compound.

¹H-NMR (CDCl₃): δ=6.14 (1H), 9.92 (2H) ppm.

Intermediate Example 1 h 6-bromopyridazin-3-amine

A solution of 250 g (1.05 mol) 3,6-dibromopyridazine in 1.2 L 25%aqueous ammonia was heated to 100° C. at 11.7 bar overnight in anautoclave. After cooling, the precipitate was filtered off, washed withwater and dried to give 137 g (75%) of the title compound.

¹H-NMR (DMSO-d6): δ=6.58 (1H), 6.69 (2H), 7.41 (1H) ppm.

Intermediate Example 1iN-cyclopropyl-2-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzamide

To a solution of 260 g (1.02 mol)4-bromo-N-cyclopropyl-2-methylbenzamide which was prepared according tointermediate example 1j in 2 L dioxane at 23° C. were added 390 gbis-(pinacolato)-diboron, 19.5 g2-dicyclohexylphosphino-2′,4′,6′-triisopropylbiphenyl, 150.g potassiumacetate and 9.37 g tris-(dibenzylidenaceton)-dipalladium(0) and themixture was refluxed for 6 h. After cooling to 23° C., water and ethylacetate were added and the mixture stirred for 15 min. The organic phasewas washed with water, dried over sodium sulfate, filtered andevaporated. The residue was purified by chromatography to give 308 g(56%) of the title compound.

¹H-NMR (300 MHz, CDCl₃): δ=0.59 (2H), 0.85 (2H), 1.33 (6H), 2.41 (3H),2.87 (1H), 5.94 (1H), 7.28 (1H), 7.60 (1H), 7.63 (1H) ppm.

Intermediate Example 1j 4-Bromo-N-cyclopropyl-2-methylbenzamide

To a stirred solution of 300 g (1.4 mol) 4-bromo-2-methylbenzoic acid in8.4 L dichloromethane at 23° C. were added 79.6 g cyclopropanamine and320.9 g EDC. After stirring overnight, the solution was washed withwater and the aqueous phase was extracted with dichloromethane. Thecombined organic phases were dried over sodium sulfate, filtered andevaporated. The remaining solid was triturated with diisopropyl ether,filtered, washed and dried in vaccuo to yield 260 g (73%) of the titlecompound.

Example 2N-cyclopropyl-4-{6-(2,3-difluoro-4-methoxyphenoxy)-8-[(3,3,3-trifluoropropyl)amino]imidazo[1,2-b]pyridazin-3-yl}-2-methylbenzamide

A solution of 31.9 g (199 mmol) 2,3-difluoro-4-methoxyphenol in 450 mLof dimethylsulfoxide was treated with 7.96 g (199 mmol) of sodiumhydride and stirred at room temperature for 1 hour. Then 16 g (33.2mmol) of4-{6-bromo-8-[(3,3,3-trifluoropropyl)amino]imidazo[1,2-b]pyridazin-3-yl}-N-cyclopropyl-2-methylbenzamide,which was prepared according to intermediate example 2a, was added andthe mixture was heated overnight at 130° C. After cooling, 300 mL ethylacetate were added and the organic phase is washed with water. Afterevaporation of the organic phase, the residue was triturated with 200 mLEtOH to give 12.05 g (65%) of the title compound.

¹H-NMR (DMSO-d6): δ=0.47-0.53 (2H), 0.62-0.70 (2H), 2.11 (3H), 2.72(2H), 2.80 (1H), 3.64 (2H), 3.92 (3H), 6.22 (1H), 7.12 (1H), 7.18 (1H),7.27 (1H), 7.63 (1H), 7.72 (1H), 7.75-7.81 (1H), 7.97 (1H), 8.24 (1H)ppm.

Intermediate Example 2a4-{6-Bromo-8-[(3,3,3-trifluoropropyl)amino]imidazo[1,2-b]pyridazin-3-yl}-N-cyclopropyl-2-methylbenzamide

A mixture comprising 127 g (292 mmol)6-bromo-3-iodo-N-(3,3,3-trifluoropropyl)imidazo[1,2-b]pyridazin-8-aminewhich was prepared according to intermediate example 2b, 95.93 g (438mmol) [4-(cyclopropylcarbamoyl)-3-methylphenyl]boronic acid which wasprepared according to intermediate example 2c, 23.8 g (29 mmol)(1,1,-bis(diphenylphosphino)ferrocene)-dichloropalladium (II), 438 mLaqueous 1M potassium carbonate solution and 973 mL tetrahydrofuran wasstirred at 80° C. for 8 hours and further 6 days at 60° C. Ethyl acetatewas added to the separated organic phase and the mixture was washed withwater. After filtration over ALLOX, the organic phase was evaporated andthe residue was triturated with 200 mL EtOH to give 71.2 g (51%) of thetitle compound.

¹H-NMR (DMSO-d6): δ=0.48-0.58 (2H), 0.63-0.73 (2H), 2.38 (3H), 2.68(2H), 2.83 (1H), 3.61 (2H), 6.49 (1H), 7.40 (1H), 7.87 (1H), 7.93 (1H),7.96-8.04 (2H), 8.32 (1H) ppm.

Intermediate Example 2b6-Bromo-3-iodo-N-(3,3,3-trifluoropropyl)imidazo[1,2-b]pyridazin-8-amine

To a solution of 119 g 295 mmol)6,8-dibromo-3-iodoimidazo[1,2-b]pyridazine which was prepared accordingto intermediate example 1e in 800 mL THF were added 66.8 g (590.8 mmol)3,3,3-trifluoropropan-1-amine and the mixture was stirred at 80° C. for2 h and at 50° C. overnight. The solution was evaporated, 600 mL ethylacetate were added and the mixture was washed with water. The organicphase was dried and evaporated to give 127 g (99%) of the titlecompound.

¹H-NMR (DMSO-d6): δ=2.63 (2H), 3.55 (2H), 6.43 (1H), 7.59 (1H),7.89-7.98 (1H) ppm.

Intermediate Example 2c [4-(cyclopropylcarbamoyl)-3-methylphenyl]boronicacid

To a solution ofN-cyclopropyl-2-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzamide(20.2 g, 67.13 mol) which was prepared according to intermediate example1i in acetone (300 mL) at rt was added sodium periodate (43.1 g, 201.40mol) and ammonium acetate (134.26 mol, 134 mL 1M aqueous solution) andthe mixture was stirred for 3 h. More water was added (120 mL), and themixture was stirred at 40° C. for 2 h more. After addition of 4 N HCl(32 mL), the organic phase was removed in vaccuo and the reminder wasextracted with ethyl actate. The organic phase was washed with sat.sodium chloride solution, filtered through a Whatman filter andevaporated. The residue was redissolved in toluene and evaporated (twotimes) to yield 14.59 g (94.3%)[4-(cyclopropylcarbamoyl)-3-methylphenyl]boronic acid: ¹H-NMR (300 MHz,d₆-DMSO): δ=8.21 (1H), 8.04 (2H), 7.56 (2H), 7.17 (1H), 2.77 (1H), 2.25(3H), 0.62 (2H), 0.47 (2H) ppm.

Example 3N-cyclopropyl-4-{6-(2,3-difluoro-4-methoxyphenoxy)-8-[(tetrahydro-2H-pyran-4-ylmethyl)amino]imidazo[1,2-b]pyridazin-3-yl}-2-methylbenzamide

To a solution of 52 mg (0.1 mmol)N-cyclopropyl-4-[6-(2,3-difluoro-4-methoxyphenoxy)-8-(methylsulfonyl)imidazo[1,2-b]pyridazin-3-yl]-2-methylbenzamidewhich was prepared according to intermediate example 3a in NMP (2 mL) atrt was 1-(tetrahydro-2H-pyran-4-yl)methanamine (35 mg, 0.3 mmol) andDIPEA (0.3 mmol, 51 μL) and the mixture was stirred at 110° C. for 72 hto give after HPLC purification 26.9 mg (47%) of the title compound

¹H-NMR (DMSO-d6): δ=0.43-0.50 (2H), 0.58-0.69 (2H), 1.22 (2H), 1.62(2H), 1.86-2.02 (1H), 2.04-2.11 (3H), 2.77 (1H), 3.19-3.30 (4H), 3.82(2H), 3.89 (3H), 6.16 (1H), 7.03-7.13 (1H), 7.15 (1H), 7.20-7.30 (1H),7.57-7.63 (1H), 7.69 (1H), 7.80 (1H), 7.93 (1H), 8.24 (1H) ppm.

Intermediate Example 3aN-cyclopropyl-4-[6-(2,3-difluoro-4-methoxyphenoxy)-8-(methylsulfonyl)imidazo[1,2-b]pyridazin-3-yl]-2-methylbenzamide

986 mg (1986 μmol)N-cyclopropyl-4-[6-(2,3-difluoro-4-methoxyphenoxy)-8-(methylsulfanyl)imidazo[1,2-b]pyridazin-3-yl]-2-methylbenzamidewhich was prepared according to intermediate example 3b were transformedin analogy to intermediate example 1b to give after working up 1040 mg(99%) of the title compound.

¹H-NMR (DMSO-d6): δ=0.44-0.51 (2H), 0.61-0.69 (2H), 2.11 (3H), 2.73-2.84(1H), 3.66 (3H), 3.91 (3H), 7.13-7.21 (1H), 7.23 (1H), 7.33-7.42 (1H),7.64-7.70 (3H), 8.30 (1H), 8.40 (1H) ppm.

Intermediate Example 3bN-cyclopropyl-4-[6-(2,3-difluoro-4-methoxyphenoxy)-8-(methylsulfanyl)imidazo[1,2-b]pyridazin-3-yl]-2-methylbenzamide

3.1 g (7428 μmol)4-[6-bromo-8-(methylsulfanyl)imidazo[1,2-b]pyridazin-3-yl]-N-cyclopropyl-2-methylbenzamidewhich was prepared according to intermediate example 1b were transformedin analogy to example 2 using 2,3-difluoro-4-methoxyphenol to give afterworking up and purification 1010 mg (27%) of the title compound.

¹H-NMR (DMSO-d6): δ=0.44-0.50 (2H), 0.59-0.70 (2H), 2.09 (3H), 2.67(3H), 2.73-2.83 (1H), 3.90 (3H), 7.06 (1H), 7.14 (1H), 7.19 (1H), 7.31(1H), 7.58-7.65 (1H), 7.67 (1H), 8.09 (1H), 8.26 (1H) ppm.

The set of compounds given in Table 1 surprisingly exhibits a superioroverall profile with respect to Mps-related activity in a functionalassay (Spindle Assembly Checkpoint Assay), antiproliferative activity(Proliferation Assay with HeLa cells), metabolic stability (in vitrometabolic stability in rat hepatocytes) and drug-drug interactionpotential (inhibition of liver enzyme CYP3A4). To further assessirreversible CYP3A4 inhibition potential, the partion ratio of thecompounds was determined, which is indicates how many substrateturnovers can be made by the enzyme prior to inactivation.

Selection criteria were Activity in Spindle Assembly Checkpoint Assay<1.0 nM, Activity in Proliferation Assay with HeLa cells <25 nM, invitro metabolic stability in rat hepatocytes Fmax ≧39% and inhibition ofliver enzyme CYP3A4 ≧5 μM, and a partion ratio ≧10.

In Table 1 Ref. is a reference compound disclosed in example 80 ofWO2012/032031A1:(N-cyclopropyl-4-{6-(3-fluorophenoxy)-8-[(3,3,3-trifluoropropyl)amino]imidazo[1,2-b]pyridazin-3-yl}-2-methylbenzamide).The compound Ref. is structurally very similar to the compounds of thepresent invention, in particular to the compound of formula (B). Despitethe structural similarity the compounds of the present invention showsuperior overall profile. From the teaching of WO2012/032031A1 one ofordinary skill in the art is not able to derive the superior profile ofthe compounds of the present invention.

TABLE 1 Fmax CYP3A4 SAC HeLa rat hep CYP3A4 partition Example [nM] [nM][%] [μM] ratio 1 0.97 2.9 44 >5 105 2 0.62 7.8 72 >10 18 3 0.08 18.139 >10 33 Ref. 0.52 23 38 >10 3Pharmaceutical Compositions of the Compounds of the Invention

This invention also relates to pharmaceutical compositions containingone or more compounds of the present invention. These compositions canbe utilised to achieve the desired pharmacological effect byadministration to a patient in need thereof. A patient, for the purposeof this invention, is a mammal, including a human, in need of treatmentfor the particular condition or disease. Therefore, the presentinvention includes pharmaceutical compositions that are comprised of apharmaceutically acceptable carrier and a pharmaceutically effectiveamount of a compound, or salt thereof, of the present invention. Apharmaceutically acceptable carrier is preferably a carrier that isrelatively non-toxic and innocuous to a patient at concentrationsconsistent with effective activity of the active ingredient so that anyside effects ascribable to the carrier do not vitiate the beneficialeffects of the active ingredient. A pharmaceutically effective amount ofcompound is preferably that amount which produces a result or exerts aninfluence on the particular condition being treated. The compounds ofthe present invention can be administered withpharmaceutically-acceptable carriers well known in the art using anyeffective conventional dosage unit forms, including immediate, slow andtimed release preparations, orally, parenterally, topically, nasally,ophthalmically, optically, sublingually, rectally, vaginally, and thelike.

For oral administration, the compounds can be formulated into solid orliquid preparations such as capsules, pills, tablets, troches, lozenges,melts, powders, solutions, suspensions, or emulsions, and may beprepared according to methods known to the art for the manufacture ofpharmaceutical compositions. The solid unit dosage forms can be acapsule that can be of the ordinary hard- or soft-shelled gelatine typecontaining, for example, surfactants, lubricants, and inert fillers suchas lactose, sucrose, calcium phosphate, and corn starch.

In another embodiment, the compounds of this invention may be tabletedwith conventional tablet bases such as lactose, sucrose and cornstarchin combination with binders such as acacia, corn starch or gelatine,disintegrating agents intended to assist the break-up and dissolution ofthe tablet following administration such as potato starch, alginic acid,corn starch, and guar gum, gum tragacanth, acacia, lubricants intendedto improve the flow of tablet granulation and to prevent the adhesion oftablet material to the surfaces of the tablet dies and punches, forexample talc, stearic acid, or magnesium, calcium or zinc stearate,dyes, colouring agents, and flavouring agents such as peppermint, oil ofwintergreen, or cherry flavouring, intended to enhance the aestheticqualities of the tablets and make them more acceptable to the patient.Suitable excipients for use in oral liquid dosage forms includedicalcium phosphate and diluents such as water and alcohols, forexample, ethanol, benzyl alcohol, and polyethylene alcohols, either withor without the addition of a pharmaceutically acceptable surfactant,suspending agent or emulsifying agent. Various other materials may bepresent as coatings or to otherwise modify the physical form of thedosage unit. For instance tablets, pills or capsules may be coated withshellac, sugar or both.

Dispersible powders and granules are suitable for the preparation of anaqueous suspension. They provide the active ingredient in admixture witha dispersing or wetting agent, a suspending agent and one or morepreservatives. Suitable dispersing or wetting agents and suspendingagents are exemplified by those already mentioned above. Additionalexcipients, for example those sweetening, flavouring and colouringagents described above, may also be present.

The pharmaceutical compositions of this invention may also be in theform of oil-in-water emulsions. The oily phase may be a vegetable oilsuch as liquid paraffin or a mixture of vegetable oils. Suitableemulsifying agents may be (1) naturally occurring gums such as gumacacia and gum tragacanth, (2) naturally occurring phosphatides such assoy bean and lecithin, (3) esters or partial esters derived form fattyacids and hexitol anhydrides, for example, sorbitan monooleate, (4)condensation products of said partial esters with ethylene oxide, forexample, polyoxyethylene sorbitan monooleate. The emulsions may alsocontain sweetening and flavouring agents.

Oily suspensions may be formulated by suspending the active ingredientin a vegetable oil such as, for example, arachis oil, olive oil, sesameoil or coconut oil, or in a mineral oil such as liquid paraffin. Theoily suspensions may contain a thickening agent such as, for example,beeswax, hard paraffin, or cetyl alcohol. The suspensions may alsocontain one or more preservatives, for example, ethyl or n-propylp-hydroxybenzoate; one or more colouring agents; one or more flavouringagents; and one or more sweetening agents such as sucrose or saccharin.

Syrups and elixirs may be formulated with sweetening agents such as, forexample, glycerol, propylene glycol, sorbitol or sucrose. Suchformulations may also contain a demulcent, and preservative, such asmethyl and propyl parabens and flavouring and colouring agents.

The compounds of this invention may also be administered parenterally,that is, subcutaneously, intravenously, intraocularly, intrasynovially,intramuscularly, or interperitoneally, as injectable dosages of thecompound in preferably a physiologically acceptable diluent with apharmaceutical carrier which can be a sterile liquid or mixture ofliquids such as water, saline, aqueous dextrose and related sugarsolutions, an alcohol such as ethanol, isopropanol, or hexadecylalcohol, glycols such as propylene glycol or polyethylene glycol,glycerol ketals such as 2,2-dimethyl-1,1-dioxolane-4-methanol, etherssuch as poly(ethylene glycol) 400, an oil, a fatty acid, a fatty acidester or, a fatty acid glyceride, or an acetylated fatty acid glyceride,with or without the addition of a pharmaceutically acceptable surfactantsuch as a soap or a detergent, suspending agent such as pectin,carbomers, methycellulose, hydroxypropylmethylcellulose, orcarboxymethylcellulose, or emulsifying agent and other pharmaceuticaladjuvants.

Illustrative of oils which can be used in the parenteral formulations ofthis invention are those of petroleum, animal, vegetable, or syntheticorigin, for example, peanut oil, soybean oil, sesame oil, cottonseedoil, corn oil, olive oil, petrolatum and mineral oil. Suitable fattyacids include oleic acid, stearic acid, isostearic acid and myristicacid. Suitable fatty acid esters are, for example, ethyl oleate andisopropyl myristate. Suitable soaps include fatty acid alkali metal,ammonium, and triethanolamine salts and suitable detergents includecationic detergents, for example dimethyl dialkyl ammonium halides,alkyl pyridinium halides, and alkylamine acetates; anionic detergents,for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether,and monoglyceride sulfates, and sulfosuccinates; non-ionic detergents,for example, fatty amine oxides, fatty acid alkanolamides, andpoly(oxyethylene-oxypropylene)s or ethylene oxide or propylene oxidecopolymers; and amphoteric detergents, for example,alkyl-beta-aminopropionates, and 2-alkylimidazoline quarternary ammoniumsalts, as well as mixtures.

The parenteral compositions of this invention will typically containfrom about 0.5% to about 25% by weight of the active ingredient insolution. Preservatives and buffers may also be used advantageously. Inorder to minimise or eliminate irritation at the site of injection, suchcompositions may contain a non-ionic surfactant having ahydrophile-lipophile balance (HLB) preferably of from about 12 to about17. The quantity of surfactant in such formulation preferably rangesfrom about 5% to about 15% by weight. The surfactant can be a singlecomponent having the above HLB or can be a mixture of two or morecomponents having the desired HLB.

Illustrative of surfactants used in parenteral formulations are theclass of polyethylene sorbitan fatty acid esters, for example, sorbitanmonooleate and the high molecular weight adducts of ethylene oxide witha hydrophobic base, formed by the condensation of propylene oxide withpropylene glycol.

The pharmaceutical compositions may be in the form of sterile injectableaqueous suspensions. Such suspensions may be formulated according toknown methods using suitable dispersing or wetting agents and suspendingagents such as, for example, sodium carboxymethylcellulose,methylcellulose, hydroxypropylmethyl-cellulose, sodium alginate,polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing orwetting agents which may be a naturally occurring phosphatide such aslecithin, a condensation product of an alkylene oxide with a fatty acid,for example, polyoxyethylene stearate, a condensation product ofethylene oxide with a long chain aliphatic alcohol, for example,heptadeca-ethyleneoxycetanol, a condensation product of ethylene oxidewith a partial ester derived form a fatty acid and a hexitol such aspolyoxyethylene sorbitol monooleate, or a condensation product of anethylene oxide with a partial ester derived from a fatty acid and ahexitol anhydride, for example polyoxyethylene sorbitan monooleate.

The sterile injectable preparation may also be a sterile injectablesolution or suspension in a non-toxic parenterally acceptable diluent orsolvent. Diluents and solvents that may be employed are, for example,water, Ringer's solution, isotonic sodium chloride solutions andisotonic glucose solutions. In addition, sterile fixed oils areconventionally employed as solvents or suspending media. For thispurpose, any bland, fixed oil may be employed including synthetic mono-or diglycerides. In addition, fatty acids such as oleic acid can be usedin the preparation of injectables.

A composition of the invention may also be administered in the form ofsuppositories for rectal administration of the drug. These compositionscan be prepared by mixing the drug with a suitable non-irritationexcipient which is solid at ordinary temperatures but liquid at therectal temperature and will therefore melt in the rectum to release thedrug. Such materials are, for example, cocoa butter and polyethyleneglycol.

Another formulation employed in the methods of the present inventionemploys transdermal delivery devices (“patches”). Such transdermalpatches may be used to provide continuous or discontinuous infusion ofthe compounds of the present invention in controlled amounts. Theconstruction and use of transdermal patches for the delivery ofpharmaceutical agents is well known in the art (see, e.g., U.S. Pat. No.5,023,252, issued Jun. 11, 1991, incorporated herein by reference). Suchpatches may be constructed for continuous, pulsatile, or on demanddelivery of pharmaceutical agents.

Controlled release formulations for parenteral administration includeliposomal, polymeric microsphere and polymeric gel formulations that areknown in the art.

It may be desirable or necessary to introduce the pharmaceuticalcomposition to the patient via a mechanical delivery device. Theconstruction and use of mechanical delivery devices for the delivery ofpharmaceutical agents is well known in the art. Direct techniques for,for example, administering a drug directly to the brain usually involveplacement of a drug delivery catheter into the patient's ventricularsystem to bypass the blood-brain barrier. One such implantable deliverysystem, used for the transport of agents to specific anatomical regionsof the body, is described in U.S. Pat. No. 5,011,472, issued Apr. 30,1991.

The compositions of the invention can also contain other conventionalpharmaceutically acceptable compounding ingredients, generally referredto as carriers or diluents, as necessary or desired. Conventionalprocedures for preparing such compositions in appropriate dosage formscan be utilized. Such ingredients and procedures include those describedin the following references, each of which is incorporated herein byreference: Powell, M. F. et al., “Compendium of Excipients forParenteral Formulations” PDA Journal of Pharmaceutical Science &Technology 1998, 52(5), 238-311; Strickley, R. G “ParenteralFormulations of Small Molecule Therapeutics Marketed in the UnitedStates (1999)— Part-1” PDA Journal of Pharmaceutical Science &Technology 1999, 53(6), 324-349; and Nema, S. et al., “Excipients andTheir Use in Injectable Products” PDA Journal of Pharmaceutical Science& Technology 1997, 51(4), 166-171.

Commonly used pharmaceutical ingredients that can be used as appropriateto formulate the composition for its intended route of administrationinclude:

acidifying agents (examples include but are not limited to acetic acid,citric acid, fumaric acid, hydrochloric acid, nitric acid);

alkalinizing agents (examples include but are not limited to ammoniasolution, ammonium carbonate, diethanolamine, monoethanolamine,potassium hydroxide, sodium borate, sodium carbonate, sodium hydroxide,triethanolamine, trolamine);

adsorbents (examples include but are not limited to powdered celluloseand activated charcoal);

aerosol propellants (examples include but are not limited to carbondioxide, CCl₂F₂, F₂ClC—CClF₂ and CClF₃)

air displacement agents (examples include but are not limited tonitrogen and argon);

antifungal preservatives (examples include but are not limited tobenzoic acid, butylparaben, ethylparaben, methylparaben, propylparaben,sodium benzoate);

antimicrobial preservatives (examples include but are not limited tobenzalkonium chloride, benzethonium chloride, benzyl alcohol,cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol,phenylmercuric nitrate and thimerosal);

antioxidants (examples include but are not limited to ascorbic acid,ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene,hypophosphorus acid, monothioglycerol, propyl gallate, sodium ascorbate,sodium bisulfite, sodium formaldehyde sulfoxylate, sodiummetabisulfite);binding materials (examples include but are not limited to blockpolymers, natural and synthetic rubber, polyacrylates, polyurethanes,silicones, polysiloxanes and styrene-butadiene copolymers);buffering agents (examples include but are not limited to potassiummetaphosphate, dipotassium phosphate, sodium acetate, sodium citrateanhydrous and sodium citrate dihydrate)carrying agents (examples include but are not limited to acacia syrup,aromatic syrup, aromatic elixir, cherry syrup, cocoa syrup, orangesyrup, syrup, corn oil, mineral oil, peanut oil, sesame oil,bacteriostatic sodium chloride injection and bacteriostatic water forinjection)chelating agents (examples include but are not limited to edetatedisodium and edetic acid)colourants (examples include but are not limited to FD&C Red No. 3, FD&CRed No. 20, FD&C Yellow No. 6, FD&C Blue No. 2, D&C Green No. 5, D&COrange No. 5, D&C Red No. 8, caramel and ferric oxide red);clarifying agents (examples include but are not limited to bentonite);emulsifying agents (examples include but are not limited to acacia,cetomacrogol, cetyl alcohol, glyceryl monostearate, lecithin, sorbitanmonooleate, polyoxyethylene 50 monostearate);encapsulating agents (examples include but are not limited to gelatinand cellulose acetate phthalate)flavourants (examples include but are not limited to anise oil, cinnamonoil, cocoa, menthol, orange oil, peppermint oil and vanillin);humectants (examples include but are not limited to glycerol, propyleneglycol and sorbitol);levigating agents (examples include but are not limited to mineral oiland glycerin);oils (examples include but are not limited to arachis oil, mineral oil,olive oil, peanut oil, sesame oil and vegetable oil);ointment bases (examples include but are not limited to lanolin,hydrophilic ointment, polyethylene glycol ointment, petrolatum,hydrophilic petrolatum, white ointment, yellow ointment, and rose waterointment);penetration enhancers (transdermal delivery) (examples include but arenot limited to monohydroxy or polyhydroxy alcohols, mono- or polyvalentalcohols, saturated or unsaturated fatty alcohols, saturated orunsaturated fatty esters, saturated or unsaturated dicarboxylic acids,essential oils, phosphatidyl derivatives, cephalin, terpenes, amides,ethers, ketones and ureas)plasticizers (examples include but are not limited to diethyl phthalateand glycerol);solvents (examples include but are not limited to ethanol, corn oil,cottonseed oil, glycerol, isopropanol, mineral oil, oleic acid, peanutoil, purified water, water for injection, sterile water for injectionand sterile water for irrigation);stiffening agents (examples include but are not limited to cetylalcohol, cetyl esters wax, microcrystalline wax, paraffin, stearylalcohol, white wax and yellow wax);suppository bases (examples include but are not limited to cocoa butterand polyethylene glycols (mixtures));surfactants (examples include but are not limited to benzalkoniumchloride, nonoxynol 10, oxtoxynol 9, polysorbate 80, sodium laurylsulfate and sorbitan mono-palmitate);suspending agents (examples include but are not limited to agar,bentonite, carbomers, carboxymethylcellulose sodium, hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose,kaolin, methylcellulose, tragacanth and veegum);sweetening agents (examples include but are not limited to aspartame,dextrose, glycerol, mannitol, propylene glycol, saccharin sodium,sorbitol and sucrose);tablet anti-adherents (examples include but are not limited to magnesiumstearate and talc);tablet binders (examples include but are not limited to acacia, alginicacid, carboxymethylcellulose sodium, compressible sugar, ethylcellulose,gelatin, liquid glucose, methylcellulose, non-crosslinked polyvinylpyrrolidone, and pregelatinized starch);tablet and capsule diluents (examples include but are not limited todibasic calcium phosphate, kaolin, lactose, mannitol, microcrystallinecellulose, powdered cellulose, precipitated calcium carbonate, sodiumcarbonate, sodium phosphate, sorbitol and starch);tablet coating agents (examples include but are not limited to liquidglucose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropylmethylcellulose, methylcellulose, ethylcellulose, cellulose acetatephthalate and shellac);tablet direct compression excipients (examples include but are notlimited to dibasic calcium phosphate);tablet disintegrants (examples include but are not limited to alginicacid, carboxymethylcellulose calcium, microcrystalline cellulose,polacrillin potassium, cross-linked polyvinylpyrrolidone, sodiumalginate, sodium starch glycollate and starch);tablet glidants (examples include but are not limited to colloidalsilica, corn starch and talc);tablet lubricants (examples include but are not limited to calciumstearate, magnesium stearate, mineral oil, stearic acid and zincstearate);tablet/capsule opaquants (examples include but are not limited totitanium dioxide);tablet polishing agents (examples include but are not limited to carnubawax and white wax);thickening agents (examples include but are not limited to beeswax,cetyl alcohol and paraffin);tonicity agents (examples include but are not limited to dextrose andsodium chloride);viscosity increasing agents (examples include but are not limited toalginic acid, bentonite, carbomers, carboxymethylcellulose sodium,methylcellulose, polyvinyl pyrrolidone, sodium alginate and tragacanth);andwetting agents (examples include but are not limited toheptadecaethylene oxycetanol, Lecithins, sorbitol monooleate,polyoxyethylene sorbitol monooleate, and polyoxyethylene stearate).

Pharmaceutical compositions according to the present invention can beillustrated as follows:

Sterile IV Solution:

A 5 mg/mL solution of the desired compound of this invention can be madeusing sterile, injectable water, and the pH is adjusted if necessary.The solution is diluted for administration to 1-2 mg/mL with sterile 5%dextrose and is administered as an IV infusion over about 60 minutes.

Lyophilised Powder for IV Administration:

A sterile preparation can be prepared with (i) 100-1000 mg of thedesired compound of this invention as a lyophilised powder, (ii) 32-327mg/mL sodium citrate, and (iii) 300-3000 mg Dextran 40. The formulationis reconstituted with sterile, injectable saline or dextrose 5% to aconcentration of 10 to 20 mg/mL, which is further diluted with saline ordextrose 5% to 0.2-0.4 mg/mL, and is administered either IV bolus or byIV infusion over 15-60 minutes.

Intramuscular Suspension:

The following solution or suspension can be prepared, for intramuscularinjection:

50 mg/mL of the desired, water-insoluble compound of this invention

5 mg/mL sodium carboxymethylcellulose

4 mg/mL TWEEN 80

9 mg/mL sodium chloride

9 mg/mL benzyl alcohol

Hard Shell Capsules:

A large number of unit capsules are prepared by filling standardtwo-piece hard galantine capsules each with 100 mg of powdered activeingredient, 150 mg of lactose, 50 mg of cellulose and 6 mg of magnesiumstearate.

Soft Gelatin Capsules:

A mixture of active ingredient in a digestible oil such as soybean oil,cottonseed oil or olive oil is prepared and injected by means of apositive displacement pump into molten gelatin to form soft gelatincapsules containing 100 mg of the active ingredient. The capsules arewashed and dried. The active ingredient can be dissolved in a mixture ofpolyethylene glycol, glycerin and sorbitol to prepare a water misciblemedicine mix.

Tablets:

A large number of tablets are prepared by conventional procedures sothat the dosage unit is 100 mg of active ingredient, 0.2 mg. ofcolloidal silicon dioxide, 5 mg of magnesium stearate, 275 mg ofmicrocrystalline cellulose, 11 mg. of starch, and 98.8 mg of lactose.Appropriate aqueous and non-aqueous coatings may be applied to increasepalatability, improve elegance and stability or delay absorption.

Immediate Release Tablets/Capsules:

These are solid oral dosage forms made by conventional and novelprocesses. These units are taken orally without water for immediatedissolution and delivery of the medication. The active ingredient ismixed in a liquid containing ingredient such as sugar, gelatin, pectinand sweeteners. These liquids are solidified into solid tablets orcaplets by freeze drying and solid state extraction techniques. The drugcompounds may be compressed with viscoelastic and thermoelastic sugarsand polymers or effervescent components to produce porous matricesintended for immediate release, without the need of water.

Combination Therapies

The compounds of this invention can be administered as the solepharmaceutical agent or in combination with one or more otherpharmaceutical agents where the combination causes no unacceptableadverse effects. The present invention relates also to suchcombinations. For example, the compounds of this invention can becombined with known anti-hyper-proliferative or other indication agents,and the like, as well as with admixtures and combinations thereof. Otherindication agents include, but are not limited to, anti-angiogenicagents, mitotic inhibitors, alkylating agents, anti-metabolites,DNA-intercalating antibiotics, growth factor inhibitors, cell cycleinhibitors, enzyme inhibitors, toposisomerase inhibitors, biologicalresponse modifiers, or anti-hormones.

The additional pharmaceutical agent can be 131I-chTNT, abarelix,abiraterone, aclarubicin, aldesleukin, alemtuzumab, alitretinoin,altretamine, aminoglutethimide, amrubicin, amsacrine, anastrozole,arglabin, arsenic trioxide, asparaginase, azacitidine, basiliximab, BAY80-6946, BAY 1000394, BAY 86-9766 (RDEA 119), belotecan, bendamustine,bevacizumab, bexarotene, bicalutamide, bisantrene, bleomycin,bortezomib, buserelin, busulfan, cabazitaxel, calcium folinate, calciumlevofolinate, capecitabine, carboplatin, carmofur, carmustine,catumaxomab, celecoxib, celmoleukin, cetuximab, chlorambucil,chlormadinone, chlormethine, cisplatin, cladribine, clodronic acid,clofarabine, crisantaspase, cyclophosphamide, cyproterone, cytarabine,dacarbazine, dactinomycin, darbepoetin alfa, dasatinib, daunorubicin,decitabine, degarelix, denileukin diftitox, denosumab, deslorelin,dibrospidium chloride, docetaxel, doxifluridine, doxorubicin,doxorubicin+estrone, eculizumab, edrecolomab, elliptinium acetate,eltrombopag, endostatin, enocitabine, epirubicin, epitiostanol, epoetinalfa, epoetin beta, eptaplatin, eribulin, erlotinib, estradiol,estramustine, etoposide, everolimus, exemestane, fadrozole, filgrastim,fludarabine, fluorouracil, flutamide, formestane, fotemustine,fulvestrant, gallium nitrate, ganirelix, gefitinib, gemcitabine,gemtuzumab, glutoxim, goserelin, histamine dihydrochloride, histrelin,hydroxycarbamide, I-125 seeds, ibandronic acid, ibritumomab tiuxetan,idarubicin, ifosfamide, imatinib, imiquimod, improsulfan, interferonalfa, interferon beta, interferon gamma, ipilimumab, irinotecan,ixabepilone, Lanreotide, lapatinib, lenalidomide, lenograstim, lentinan,letrozole, Leuprorelin, levamisole, lisuride, lobaplatin, lomustine,lonidamine, masoprocol, medroxyprogesterone, megestrol, melphalan,mepitiostane, mercaptopurine, methotrexate, methoxsalen, Methylaminolevulinate, methyltestosterone, mifamurtide, miltefosine,miriplatin, mitobronitol, mitoguazone, mitolactol, mitomycin, mitotane,mitoxantrone, nedaplatin, nelarabine, nilotinib, nilutamide,nimotuzumab, nimustine, nitracrine, ofatumumab, omeprazole, oprelvekin,oxaliplatin, p53 gene therapy, paclitaxel, palifermin, palladium-103seed, pamidronic acid, panitumumab, pazopanib, pegaspargase, PEG-epoetinbeta (methoxy PEG-epoetin beta), pegfilgrastim, peginterferon alfa-2b,pemetrexed, pentazocine, pentostatin, peplomycin, perfosfamide,picibanil, pirarubicin, plerixafor, plicamycin, poliglusam,polyestradiol phosphate, polysaccharide-K, porfimer sodium,pralatrexate, prednimustine, procarbazine, quinagolide, raloxifene,raltitrexed, ranimustine, razoxane, regorafenib, risedronic acid,rituximab, romidepsin, romiplostim, sargramostim, sipuleucel-T,sizofiran, sobuzoxane, sodium glycididazole, sorafenib, streptozocin,sunitinib, talaporfin, tamibarotene, tamoxifen, tasonermin, teceleukin,tegafur, tegafur+gimeracil+oteracil, temoporfin, temozolomide,temsirolimus, teniposide, testosterone, tetrofosmin, thalidomide,thiotepa, thymalfasin, tioguanine, tocilizumab, topotecan, toremifene,tositumomab, trabectedin, trastuzumab, treosulfan, tretinoin,trilostane, triptorelin, trofosfamide, tryptophan, ubenimex, valrubicin,vandetanib, vapreotide, vemurafenib, vinblastine, vincristine,vindesine, vinflunine, vinorelbine, vorinostat, vorozole, yttrium-90glass microspheres, zinostatin, zinostatin stimalamer, zoledronic acid,zorubicin.

Preferably, the additional pharmaceutical agent is selected from:afinitor, aldesleukin, alendronic acid, alfaferone, alitretinoin,allopurinol, aloprim, aloxi, altretamine, aminoglutethimide, amifostine,amrubicin, amsacrine, anastrozole, anzmet, aranesp, arglabin, arsenictrioxide, aromasin, 5-azacytidine, azathioprine, BCG or tice BCG,bestatin, betamethasone acetate, betamethasone sodium phosphate,bexarotene, bleomycin sulfate, broxuridine, bortezomib, busulfan,calcitonin, campath, capecitabine, carboplatin, casodex, cefesone,celmoleukin, cerubidine, chlorambucil, cisplatin, cladribine,cladribine, clodronic acid, cyclophosphamide, cytarabine, dacarbazine,dactinomycin, DaunoXome, decadron, decadron phosphate, delestrogen,denileukin diftitox, depo-medrol, deslorelin, dexrazoxane,diethylstilbestrol, diflucan, docetaxel, doxifluridine, doxorubicin,dronabinol, DW-166HC, eligard, elitek, ellence, emend, epirubicin,epoetin alfa, epogen, eptaplatin, ergamisol, estrace, estradiol,estramustine phosphate sodium, ethinyl estradiol, ethyol, etidronicacid, etopophos, etoposide, fadrozole, farston, filgrastim, finasteride,fligrastim, floxuridine, fluconazole, fludarabine, 5-fluorodeoxyuridinemonophosphate, 5-fluorouracil (5-FU), fluoxymesterone, flutamide,formestane, fosteabine, fotemustine, fulvestrant, gammagard,gemcitabine, gemtuzumab, gleevec, gliadel, goserelin, granisetron HCl,histrelin, hycamtin, hydrocortone, eyrthro-hydroxynonyladenine,hydroxyurea, ibritumomab tiuxetan, idarubicin, ifosfamide, interferonalpha, interferon-alpha 2, interferon alfa-2A, interferon alfa-2B,interferon alfa-n1, interferon alfa-n3, interferon beta, interferongamma-1a, interleukin-2, intron A, iressa, irinotecan, kytril, lentinansulfate, letrozole, leucovorin, leuprolide, leuprolide acetate,Levamisole, Levofolinic acid calcium salt, levothroid, levoxyl,lomustine, lonidamine, marinol, mechlorethamine, mecobalamin,medroxyprogesterone acetate, megestrol acetate, melphalan, menest,6-mercaptopurine, Mesna, methotrexate, metvix, miltefosine, minocycline,mitomycin C, mitotane, mitoxantrone, Modrenal, Myocet, nedaplatin,neulasta, neumega, neupogen, nilutamide, nolvadex, NSC-631570, OCT-43,octreotide, ondansetron HCl, orapred, oxaliplatin, paclitaxel,pediapred, pegaspargase, Pegasys, pentostatin, picibanil, pilocarpineHCl, pirarubicin, plicamycin, porfimer sodium, prednimustine,prednisolone, prednisone, premarin, procarbazine, procrit, raltitrexed,RDEA 119, rebif, rhenium-186 etidronate, rituximab, roferon-A,romurtide, salagen, sandostatin, sargramostim, semustine, sizofiran,sobuzoxane, solu-medrol, sparfosic acid, stem-cell therapy,streptozocin, strontium-89 chloride, synthroid, tamoxifen, tamsulosin,tasonermin, tastolactone, taxotere, teceleukin, temozolomide,teniposide, testosterone propionate, testred, thioguanine, thiotepa,thyrotropin, tiludronic acid, topotecan, toremifene, tositumomab,trastuzumab, treosulfan, tretinoin, trexall, trimethylmelamine,trimetrexate, triptorelin acetate, triptorelin pamoate, UFT, uridine,valrubicin, vesnarinone, vinblastine, vincristine, vindesine,vinorelbine, virulizin, zinecard, zinostatin stimalamer, zofran,ABI-007, acolbifene, actimmune, affinitak, aminopterin, arzoxifene,asoprisnil, atamestane, atrasentan, sorafenib (BAY 43-9006), avastin,CCI-779, CDC-501, celebrex, cetuximab, crisnatol, cyproterone acetate,decitabine, DN-101, doxorubicin-MTC, dSLIM, dutasteride, edotecarin,eflornithine, exatecan, fenretinide, histamine dihydrochloride,histrelin hydrogel implant, holmium-166 DOTMP, ibandronic acid,interferon gamma, intron-PEG, ixabepilone, keyhole limpet hemocyanin,L-651582, lanreotide, lasofoxifene, libra, lonafarnib, miproxifene,minodronate, MS-209, liposomal MTP-PE, MX-6, nafarelin, nemorubicin,neovastat, nolatrexed, oblimersen, onco-TCS, osidem, paclitaxelpolyglutamate, pamidronate disodium, PN-401, QS-21, quazepam, R-1549,raloxifene, ranpirnase, 13-cis-retinoic acid, satraplatin, seocalcitol,T-138067, tarceva, taxoprexin, thymosin alpha 1, tiazofurine,tipifarnib, tirapazamine, TLK-286, toremifene, TransMID-107R, valspodar,vapreotide, vatalanib, verteporfin, vinflunine, Z-100, and zoledronicacid or combinations thereof.

Optional anti-hyper-proliferative agents which can be added to thecomposition include but are not limited to compounds listed on thecancer chemotherapy drug regimens in the 11^(th) Edition of the MerckIndex, (1996), which is hereby incorporated by reference, such asasparaginase, bleomycin, carboplatin, carmustine, chlorambucil,cisplatin, colaspase, cyclophosphamide, cytarabine, dacarbazine,dactinomycin, daunorubicin, doxorubicin (adriamycine), epirubicin,epothilone, an epothilone derivative, etoposide, 5-fluorouracil,hexamethylmelamine, hydroxyurea, ifosfamide, irinotecan, leucovorin,lomustine, mechlorethamine, 6-mercaptopurine, mesna, methotrexate,mitomycin C, mitoxantrone, prednisolone, prednisone, procarbazine,raloxifen, streptozocin, tamoxifen, thioguanine, topotecan, vinblastine,vincristine, and vindesine.

Other anti-hyper-proliferative agents suitable for use with thecomposition of the invention include but are not limited to thosecompounds acknowledged to be used in the treatment of neoplasticdiseases in Goodman and Gilman's The Pharmacological Basis ofTherapeutics (Ninth Edition), editor Molinoff et al., publ. byMcGraw-Hill, pages 1225-1287, (1996), which is hereby incorporated byreference, such as aminoglutethimide, L-asparaginase, azathioprine,5-azacytidine cladribine, busulfan, diethylstilbestrol,2′,2′-difluorodeoxycytidine, docetaxel, erythrohydroxynonyl adenine,ethinyl estradiol, 5-fluorodeoxyuridine, 5-fluorodeoxyuridinemonophosphate, fludarabine phosphate, fluoxymesterone, flutamide,hydroxyprogesterone caproate, idarubicin, interferon,medroxyprogesterone acetate, megestrol acetate, melphalan, mitotane,paclitaxel, pentostatin, N-phosphonoacetyl-L-aspartate (PALA),plicamycin, semustine, teniposide, testosterone propionate, thiotepa,trimethylmelamine, uridine, and vinorelbine.

Other anti-hyper-proliferative agents suitable for use with thecomposition of the invention include but are not limited to otheranti-cancer agents such as epothilone and its derivatives, irinotecan,raloxifen and topotecan.

The compounds of the invention may also be administered in combinationwith protein therapeutics. Such protein therapeutics suitable for thetreatment of cancer or other angiogenic disorders and for use with thecompositions of the invention include, but are not limited to, aninterferon (e.g., interferon .alpha., .beta., or .gamma.) supraagonisticmonoclonal antibodies, Tuebingen, TRP-1 protein vaccine, Colostrinin,anti-FAP antibody, YH-16, gemtuzumab, infliximab, cetuximab,trastuzumab, denileukin diftitox, rituximab, thymosin alpha 1,bevacizumab, mecasermin, mecasermin rinfabate, oprelvekin, natalizumab,rhMBL, MFE-CP1+ZD-2767-P, ABT-828, ErbB2-specific immunotoxin, SGN-35,MT-103, rinfabate, AS-1402, B43-genistein, L-19 basedradioimmunotherapeutics, AC-9301, NY-ESO-1 vaccine, IMC-1C11, CT-322,rhCC10, r(m)CRP, MORAb-009, aviscumine, MDX-1307, Her-2 vaccine,APC-8024, NGR-hTNF, rhH1.3, IGN-311, Endostatin, volociximab, PRO-1762,lexatumumab, SGN-40, pertuzumab, EMD-273063, L19-IL-2 fusion protein,PRX-321, CNTO-328, MDX-214, tigapotide, CAT-3888, Labetuzumab,alpha-particle-emitting radioisotope-llinked lintuzumab, EM-1421,HyperAcute vaccine, tucotuzumab celmoleukin, galiximab, HPV-16-E7,Javelin—prostate cancer, Javelin—melanoma, NY-ESO-1 vaccine, EGFvaccine, CYT-004-MelQbG10, WT1 peptide, oregovomab, ofatumumab,zalutumumab, cintredekin besudotox, WX-G250, Albuferon, aflibercept,denosumab, vaccine, CTP-37, efungumab, or 131I-chTNT-1/B. Monoclonalantibodies useful as the protein therapeutic include, but are notlimited to, muromonab-CD3, abciximab, edrecolomab, daclizumab,gentuzumab, alemtuzumab, ibritumomab, cetuximab, bevicizumab,efalizumab, adalimumab, omalizumab, muromomab-CD3, rituximab,daclizumab, trastuzumab, palivizumab, basiliximab, and infliximab.

The compounds of the invention may also be combined with biologicaltherapeutic agents, such as antibodies (e.g. avastin, rituxan, erbitux,herceptin), or recombinant proteins.

The compounds of the invention may also be in combination withantiangiogenesis agents, such as, for example, with avastin, axitinib,DAST, recentin, sorafenib or sunitinib. Combinations with inhibitors ofproteasomes or mTOR inhibitors, or anti-hormones or steroidal metabolicenzyme inhibitors are also possible.

Generally, the use of cytotoxic and/or cytostatic agents in combinationwith a compound or composition of the present invention will serve to:

-   (1) yield better efficacy in reducing the growth of a tumour or even    eliminate the tumour as compared to administration of either agent    alone,-   (2) provide for the administration of lesser amounts of the    administered chemotherapeutic agents,-   (3) provide for a chemotherapeutic treatment that is well tolerated    in the patient with fewer deleterious pharmacological complications    than observed with single agent chemotherapies and certain other    combined therapies,-   (4) provide for treating a broader spectrum of different cancer    types in mammals, especially humans,-   (5) provide for a higher response rate among treated patients,-   (6) provide for a longer survival time among treated patients    compared to standard chemotherapy treatments,-   (7) provide a longer time for tumour progression, and/or-   (8) yield efficacy and tolerability results at least as good as    those of the agents used alone, compared to known instances where    other cancer agent combinations produce antagonistic effects.    Methods of Sensitizing Cells to Radiation

In a distinct embodiment of the present invention, a compound of thepresent invention may be used to sensitize a cell to radiation. That is,treatment of a cell with a compound of the present invention prior toradiation treatment of the cell renders the cell more susceptible to DNAdamage and cell death than the cell would be in the absence of anytreatment with a compound of the invention. In one aspect, the cell istreated with at least one compound of the invention.

Thus, the present invention also provides a method of killing a cell,wherein a cell is administered one or more compounds of the invention incombination with conventional radiation therapy.

The present invention also provides a method of rendering a cell moresusceptible to cell death, wherein the cell is treated one or morecompounds of the invention prior to the treatment of the cell to causeor induce cell death. In one aspect, after the cell is treated with oneor more compounds of the invention, the cell is treated with at leastone compound, or at least one method, or a combination thereof, in orderto cause DNA damage for the purpose of inhibiting the function of thenormal cell or killing the cell.

In one embodiment, a cell is killed by treating the cell with at leastone DNA damaging agent. That is, after treating a cell with one or morecompounds of the invention to sensitize the cell to cell death, the cellis treated with at least one DNA damaging agent to kill the cell. DNAdamaging agents useful in the present invention include, but are notlimited to, chemotherapeutic agents (e.g., cisplatinum), ionizingradiation (X-rays, ultraviolet radiation), carcinogenic agents, andmutagenic agents.

In another embodiment, a cell is killed by treating the cell with atleast one method to cause or induce DNA damage. Such methods include,but are not limited to, activation of a cell signalling pathway thatresults in DNA damage when the pathway is activated, inhibiting of acell signalling pathway that results in DNA damage when the pathway isinhibited, and inducing a biochemical change in a cell, wherein thechange results in DNA damage. By way of a non-limiting example, a DNArepair pathway in a cell can be inhibited, thereby preventing the repairof DNA damage and resulting in an abnormal accumulation of DNA damage ina cell.

In one aspect of the invention, a compound of the invention isadministered to a cell prior to the radiation or other induction of DNAdamage in the cell. In another aspect of the invention, a compound ofthe invention is administered to a cell concomitantly with the radiationor other induction of DNA damage in the cell. In yet another aspect ofthe invention, a compound of the invention is administered to a cellimmediately after radiation or other induction of DNA damage in the cellhas begun.

In another aspect, the cell is in vitro. In another embodiment, the cellis in vivo.

As mentioned supra, the compounds of the present invention havesurprisingly been found to effectively inhibit Mps-1 and may thereforebe used for the treatment or prophylaxis of diseases of uncontrolledcell growth, proliferation and/or survival, inappropriate cellularimmune responses, or inappropriate cellular inflammatory responses, ordiseases which are accompanied with uncontrolled cell growth,proliferation and/or survival, inappropriate cellular immune responses,or inappropriate cellular inflammatory responses, particularly in whichthe uncontrolled cell growth, proliferation and/or survival,inappropriate cellular immune responses, or inappropriate cellularinflammatory responses is mediated by Mps-1, such as, for example,haematological tumours, solid tumours, and/or metastases thereof, e.g.Leukaemias and myelodysplastic syndrome, malignant lymphomas, head andneck tumours including brain tumours and brain metastases, tumours ofthe thorax including non-small cell and small cell lung tumours,gastrointestinal tumours, endocrine tumours, mammary and othergynaecological tumours, urological tumours including renal, bladder andprostate tumours, skin tumours, and sarcomas, and/or metastases thereof.

In accordance with another aspect therefore, the present inventioncovers a compound of formula (A), (B) or (C), or a stereoisomer, atautomer, an N-oxide, a hydrate, a solvate, or a salt thereof,particularly a pharmaceutically acceptable salt thereof, or a mixture ofsame, as described and defined herein, for use in the treatment orprophylaxis of a disease, as mentioned supra.

Another particular aspect of the present invention is therefore the useof a compound of formula (A), (B) or (C), described supra, or astereoisomer, a tautomer, an N-oxide, a hydrate, a solvate, or a saltthereof, particularly a pharmaceutically acceptable salt thereof, or amixture of same, for the prophylaxis or treatment of a disease.

Another particular aspect of the present invention is therefore the useof a compound of formula (A), (B) or (C) described supra formanufacturing a pharmaceutical composition for the treatment orprophylaxis of a disease.

The diseases referred to in the two preceding paragraphs are diseases ofuncontrolled cell growth, proliferation and/or survival, inappropriatecellular immune responses, or inappropriate cellular inflammatoryresponses, or diseases which are accompanied with uncontrolled cellgrowth, proliferation and/or survival, inappropriate cellular immuneresponses, or inappropriate cellular inflammatory responses,particularly in which the uncontrolled cell growth, proliferation and/orsurvival, inappropriate cellular immune responses, or inappropriatecellular inflammatory responses is mediated by Mps-1, such as, forexample, haematological tumours, solid tumours, and/or metastasesthereof, e.g. leukaemias and myelodysplastic syndrome, malignantlymphomas, head and neck tumours including brain tumours and brainmetastases, tumours of the thorax including non-small cell and smallcell lung tumours, gastrointestinal tumours, endocrine tumours, mammaryand other gynaecological tumours, urological tumours including renal,bladder and prostate tumours, skin tumours, and sarcomas, and/ormetastases thereof.

The term “inappropriate” within the context of the present invention, inparticular in the context of “inappropriate cellular immune responses,or inappropriate cellular inflammatory responses”, as used herein, is tobe understood as preferably meaning a response which is less than, orgreater than normal, and which is associated with, responsible for, orresults in, the pathology of said diseases.

Preferably, the use is in the treatment or prophylaxis of diseases,wherein the diseases are haemotological tumours, solid tumours and/ormetastases thereof.

Method of Treating Hyper-Proliferative Disorders

The present invention relates to a method for using the compounds of thepresent invention and compositions thereof, to treat mammalianhyper-proliferative disorders. Compounds can be utilized to inhibit,block, reduce, decrease, etc., cell proliferation and/or cell division,and/or produce apoptosis. This method comprises administering to amammal in need thereof, including a human, an amount of a compound ofthis invention, or a pharmaceutically acceptable salt, isomer,polymorph, metabolite, hydrate, solvate or ester thereof; etc. which iseffective to treat the disorder. Hyper-proliferative disorders includebut are not limited, e.g., psoriasis, keloids, and other hyperplasiasaffecting the skin, benign prostate hyperplasia (BPH), solid tumours,such as cancers of the breast, respiratory tract, brain, reproductiveorgans, digestive tract, urinary tract, eye, liver, skin, head and neck,thyroid, parathyroid and their distant metastases. Those disorders alsoinclude lymphomas, sarcomas, and leukaemias.

Examples of breast cancer include, but are not limited to invasiveductal carcinoma, invasive lobular carcinoma, ductal carcinoma in situ,and lobular carcinoma in situ.

Examples of cancers of the respiratory tract include, but are notlimited to small-cell and non-small-cell lung carcinoma, as well asbronchial adenoma and pleuropulmonary blastoma.

Examples of brain cancers include, but are not limited to brain stem andhypophtalmic glioma, cerebellar and cerebral astrocytoma,medulloblastoma, ependymoma, as well as neuroectodermal and pinealtumour.

Tumours of the male reproductive organs include, but are not limited toprostate and testicular cancer. Tumours of the female reproductiveorgans include, but are not limited to endometrial, cervical, ovarian,vaginal, and vulvar cancer, as well as sarcoma of the uterus.

Tumours of the digestive tract include, but are not limited to anal,colon, colorectal, oesophageal, gallbladder, gastric, pancreatic,rectal, small-intestine, and salivary gland cancers. Tumours of theurinary tract include, but are not limited to bladder, penile, kidney,renal pelvis, ureter, urethral and human papillary renal cancers.

Eye cancers include, but are not limited to intraocular melanoma andretinoblastoma.

Examples of liver cancers include, but are not limited to hepatocellularcarcinoma (liver cell carcinomas with or without fibrolamellar variant),cholangiocarcinoma (intrahepatic bile duct carcinoma), and mixedhepatocellular cholangiocarcinoma.

Skin cancers include, but are not limited to squamous cell carcinoma,Kaposi's sarcoma, malignant melanoma, Merkel cell skin cancer, andnon-melanoma skin cancer.

Head-and-neck cancers include, but are not limited to laryngeal,hypopharyngeal, nasopharyngeal, oropharyngeal cancer, lip and oralcavity cancer and squamous cell. Lymphomas include, but are not limitedto AIDS-related lymphoma, non-Hodgkin's Lymphoma, cutaneous T-cellLymphoma, Burkitt Lymphoma, Hodgkin's disease, and lymphoma of thecentral nervous system.

Sarcomas include, but are not limited to sarcoma of the soft tissue,osteosarcoma, malignant fibrous histiocytoma, lymphosarcoma, andrhabdomyosarcoma.

Leukemias include, but are not limited to acute myeloid leukemia, acutelymphoblastic leukemia, chronic lymphocytic leukemia, chronicmyelogenous leukemia, and hairy cell leukemia.

These disorders have been well characterized in humans, but also existwith a similar etiology in other mammals, and can be treated byadministering pharmaceutical compositions of the present invention.

The term “treating” or “treatment” as stated throughout this document isused conventionally, e.g., the management or care of a subject for thepurpose of combating, alleviating, reducing, relieving, improving thecondition of, etc., of a disease or disorder, such as a carcinoma.

Methods of Treating Kinase Disorders

The present invention also provides methods for the treatment ofdisorders associated with aberrant mitogen extracellular kinaseactivity, including, but not limited to stroke, heart failure,hepatomegaly, cardiomegaly, diabetes, Alzheimer's disease, cysticfibrosis, symptoms of xenograft rejections, septic shock or asthma.

Effective amounts of compounds of the present invention can be used totreat such disorders, including those diseases (e.g., cancer) mentionedin the Background section above. Nonetheless, such cancers and otherdiseases can be treated with compounds of the present invention,regardless of the mechanism of action and/or the relationship betweenthe kinase and the disorder.

The phrase “aberrant kinase activity” or “aberrant tyrosine kinaseactivity,” includes any abnormal expression or activity of the geneencoding the kinase or of the polypeptide it encodes. Examples of suchaberrant activity, include, but are not limited to, over-expression ofthe gene or polypeptide; gene amplification; mutations which produceconstitutively-active or hyperactive kinase activity; gene mutations,deletions, substitutions, additions, etc.

The present invention also provides for methods of inhibiting a kinaseactivity, especially of mitogen extracellular kinase, comprisingadministering an effective amount of a compound of the presentinvention, including salts, polymorphs, metabolites, hydrates, solvates,prodrugs (e.g.: esters) thereof, and diastereoisomeric forms thereof.Kinase activity can be inhibited in cells (e.g., in vitro), or in thecells of a mammalian subject, especially a human patient in need oftreatment.

Methods of Treating Angiogenic Disorders

The present invention also provides methods of treating disorders anddiseases associated with excessive and/or abnormal angiogenesis.

Inappropriate and ectopic expression of angiogenesis can be deleteriousto an organism. A number of pathological conditions are associated withthe growth of extraneous blood vessels. These include, e.g., diabeticretinopathy, ischemic retinal-vein occlusion, and retinopathy ofprematurity [Aiello et at. New Engl. J. Med. 1994, 331, 1480; Peer etat. Lab. Invest. 1995, 72, 638], age-related macular degeneration [AMD;see, Lopez et at. Invest. Opththalmol. Vis. Sci. 1996, 37, 855],neovascular glaucoma, psoriasis, retrolental fibroplasias, angiofibroma,inflammation, rheumatoid arthritis (RA), restenosis, in-stentrestenosis, vascular graft restenosis, etc. In addition, the increasedblood supply associated with cancerous and neoplastic tissue, encouragesgrowth, leading to rapid tumour enlargement and metastasis. Moreover,the growth of new blood and lymph vessels in a tumour provides an escaperoute for renegade cells, encouraging metastasis and the consequencespread of the cancer. Thus, compounds of the present invention can beutilized to treat and/or prevent any of the aforementioned angiogenesisdisorders, e.g., by inhibiting and/or reducing blood vessel formation;by inhibiting, blocking, reducing, decreasing, etc. endothelial cellproliferation or other types involved in angiogenesis, as well ascausing cell death or apoptosis of such cell types.

Dose and Administration

Based upon standard laboratory techniques known to evaluate compoundsuseful for the treatment of hyper-proliferative disorders and angiogenicdisorders, by standard toxicity tests and by standard pharmacologicalassays for the determination of treatment of the conditions identifiedabove in mammals, and by comparison of these results with the results ofknown medicaments that are used to treat these conditions, the effectivedosage of the compounds of this invention can readily be determined fortreatment of each desired indication. The amount of the activeingredient to be administered in the treatment of one of theseconditions can vary widely according to such considerations as theparticular compound and dosage unit employed, the mode ofadministration, the period of treatment, the age and sex of the patienttreated, and the nature and extent of the condition treated.

The total amount of the active ingredient to be administered willgenerally range from about 0.001 mg/kg to about 200 mg/kg body weightper day, and preferably from about 0.01 mg/kg to about 20 mg/kg bodyweight per day. Clinically useful dosing schedules will range from oneto three times a day dosing to once every four weeks dosing. Inaddition, “drug holidays” in which a patient is not dosed with a drugfor a certain period of time, may be beneficial to the overall balancebetween pharmacological effect and tolerability. A unit dosage maycontain from about 0.5 mg to about 1500 mg of active ingredient, and canbe administered one or more times per day or less than once a day. Theaverage daily dosage for administration by injection, includingintravenous, intramuscular, subcutaneous and parenteral injections, anduse of infusion techniques will preferably be from 0.01 to 200 mg/kg oftotal body weight. The average daily rectal dosage regimen willpreferably be from 0.01 to 200 mg/kg of total body weight. The averagedaily vaginal dosage regimen will preferably be from 0.01 to 200 mg/kgof total body weight. The average daily topical dosage regimen willpreferably be from 0.1 to 200 mg administered between one to four timesdaily. The transdermal concentration will preferably be that required tomaintain a daily dose of from 0.01 to 200 mg/kg. The average dailyinhalation dosage regimen will preferably be from 0.01 to 100 mg/kg oftotal body weight.

Of course the specific initial and continuing dosage regimen for eachpatient will vary according to the nature and severity of the conditionas determined by the attending diagnostician, the activity of thespecific compound employed, the age and general condition of thepatient, time of administration, route of administration, rate ofexcretion of the drug, drug combinations, and the like. The desired modeof treatment and number of doses of a compound of the present inventionor a pharmaceutically acceptable salt or ester or composition thereofcan be ascertained by those skilled in the art using conventionaltreatment tests.

Preferably, the diseases of said method are haematological tumours,solid tumour and/or metastases thereof.

The compounds of the present invention can be used in particular intherapy and prevention, i.e. prophylaxis, of tumour growth andmetastases, especially in solid tumours of all indications and stageswith or without pre-treatment of the tumour growth.

Methods of testing for a particular pharmacological or pharmaceuticalproperty are well known to persons skilled in the art.

The example testing experiments described herein serve to illustrate thepresent invention and the invention is not limited to the examplesgiven.

Biological assay: Proliferation Assay Cultivated tumour cells (MCF7,hormone dependent human mammary carcinoma cells, ATCC HTB22; NCI-H460,human non-small cell lung carcinoma cells, ATCC HTB-177; DU 145,hormone-independent human prostate carcinoma cells, ATCC HTB-81;HeLa-MaTu, human cervical carcinoma cells, EPO-GmbH, Berlin;HeLa-MaTu-ADR, multidrug-resistant human cervical carcinoma cells,EPO-GmbH, Berlin; HeLa human cervical tumour cells, ATCC CCL-2; B16F10mouse melanoma cells, ATCC CRL-6475) were plated at a density of 5000cells/well (MCF7, DU145, HeLa-MaTu-ADR), 3000 cells/well (NCI-H460,HeLa-MaTu, HeLa), or 1000 cells/well (B16F10) in a 96-well multititerplate in 200 μL of their respective growth medium supplemented 10% fetalcalf serum. After 24 hours, the cells of one plate (zero-point plate)were stained with crystal violet (see below), while the medium of theother plates was replaced by fresh culture medium (200 μl), to which thetest substances were added in various concentrations (0 μM, as well asin the range of 0.01-30 μM; the final concentration of the solventdimethyl sulfoxide was 0.5%). The cells were incubated for 4 days in thepresence of test substances. Cell proliferation was determined bystaining the cells with crystal violet: the cells were fixed by adding20 μl/measuring point of an 11% glutaric aldehyde solution for 15minutes at room temperature. After three washing cycles of the fixedcells with water, the plates were dried at room temperature. The cellswere stained by adding 100 μl/measuring point of a 0.1% crystal violetsolution (pH 3.0). After three washing cycles of the stained cells withwater, the plates were dried at room temperature. The dye was dissolvedby adding 100 μl/measuring point of a 10% acetic acid solution. Theextinction was determined by photometry at a wavelength of 595 nm. Thechange of cell number, in percent, was calculated by normalization ofthe measured values to the extinction values of the zero-point plate(=0%) and the extinction of the untreated (0 μm) cells (=100%). The IC50values were determined by means of a 4 parameter fit using the company'sown software.

Mps-1 Kinase Assay

The human kinase Mps-1 phosphorylates a biotinylated substrate peptide.Detection of the phosphorylated product is achieved by time-resolvedfluorescence resonance energy transfer (TR-FRET) from Europium-labelledanti-phospho-Serine/Threonine antibody as donor to streptavidin labelledwith cross-linked allophycocyanin (SA-XLent) as acceptor. Compounds aretested for their inhibition of the kinase activity.

N-terminally GST-tagged human full length recombinant Mps-1 kinase(purchased from Invitrogen, Karslruhe, Germany, cat. no PV4071) wasused. As substrate for the kinase reaction a biotinylated peptide of theamino-acid sequence PWDPDDADITEILG (C-terminus in amide form, purchasedfrom Biosynthan GmbH, Berlin) was used.

For the assay 50 nL of a 100-fold concentrated solution of the testcompound in DMSO was pipetted into a black low volume 384 wellmicrotiter plate (Greiner Bio-One, Frickenhausen, Germany), 2 μl of asolution of Mps-1 in assay buffer [0.1 mM sodium-ortho-vanadate, 10 mMMgCl₂, 2 mM DTT, 25 mM Hepes pH 7.7, 0.05% BSA, 0.001% Pluronic F-127]were added and the mixture was incubated for 15 min at 22° C. to allowpre-binding of the test compounds to Mps-1 before the start of thekinase reaction. Then the kinase reaction was started by the addition of3 μl of a solution of 16.7 adenosine-tri-phosphate (ATP, 16.7 μM=>finalconc. in the 5 μl assay volume is 10 μM) and peptide substrate (1.67μM=>final conc. in the 5 μl assay volume is 1 μM) in assay buffer andthe resulting mixture was incubated for a reaction time of 60 min at 22°C. The concentration of Mps-1 in the assay was adjusted to the activityof the enzyme lot and was chosen appropriate to have the assay in thelinear range, typical enzyme concentrations were in the range of about 1nM (final conc. in the 5 μl assay volume). The reaction was stopped bythe addition of 3 μl of a solution of HTRF detection reagents (100 mMHepes pH 7.4, 0.1% BSA, 40 mM EDTA, 140 nM Streptavidin-XLent[#61GSTXLB, Fa. Cis Biointernational, Marcoule, France], 1.5 nManti-phospho(Ser/Thr)-Europium-antibody [#AD0180, PerkinElmer LAS,Rodgau-Jügesheim, Germany].

The resulting mixture was incubated 1 h at 22° C. to allow the bindingof the phosphorylated peptide to theanti-phospho(Ser/Thr)-Europium-antibody. Subsequently the amount ofphosphorylated substrate was evaluated by measurement of the resonanceenergy transfer from the Europium-labelled anti-phospho(Ser/Thr)antibody to the Streptavidin-XLent. Therefore, the fluorescenceemissions at 620 nm and 665 nm after excitation at 350 nm was measuredin a Viewlux TR-FRET reader (PerkinElmer LAS, Rodgau-Jügesheim,Germany). The “blank-corrected normalized ratio” (a Viewlux specificreadout, similar to the traditional ratio of the emissions at 665 nm andat 622 nm, in which blank and Eu-donor crosstalk are subtracted from the665 nm signal before the ratio is calculated) was taken as the measurefor the amount of phosphorylated substrate. The data were normalised(enzyme reaction without inhibitor=0% inhibition, all other assaycomponents but no enzyme=100% inhibition). Test compounds were tested onthe same microtiter plate at 10 different concentrations in the range of20 μM to 1 nM (20 μM, 6.7 μM, 2.2 μM, 0.74 μM, 0.25 μM, 82 nM, 27 nM,9.2 nM, 3.1 nM and 1 nM, dilution series prepared before the assay atthe level of the 100 fold conc. stock solutions by serial 1:3 dilutions)in duplicate values for each concentration and IC₅₀ values werecalculated by a 4 parameter fit using an in-house software.

Spindle Assembly Checkpoint Assay

The spindle assembly checkpoint assures the proper segregation ofchromosomes during mitosis. Upon entry into mitosis, chromosomes beginto condensate which is accompanied by the phosphorylation of histone H3on serine 10. Dephosphorylation of histone H3 on serine 10 begins inanaphase and ends at early telophase. Accordingly, phosphorylation ofhistone H3 on serine 10 can be utilized as a marker of cells in mitosis.Nocodazole is a microtubule destabilizing substance. Thus, nocodazoleinterferes with microtubule dynamics and mobilises the spindle assemblycheckpoint. The cells arrest in mitosis at G2/M transition and exhibitphosphorylated histone H3 on serine 10. An inhibition of the spindleassembly checkpoint by Mps-1 inhibitors overrides the mitotic blockagein the presence of nocodazole, and the cells complete mitosisprematurely. This alteration is detected by the decrease of cells withphosphorylation of histone H3 on serine 10. This decline is used as amarker to determine the capability of compounds of the present inventionto induce a mitotic breakthrough.

Cultivated cells of the human cervical tumour cell line HeLa (ATCCCCL-2) were plated at a density of 2500 cells/well in a 384-wellmicrotiter plate in 20 μl Dulbeco's Medium (w/o phenol red, w/o sodiumpyruvate, w 1000 mg/ml glucose, w pyridoxine) supplemented with 1% (v/v)glutamine, 1% (v/v) penicillin, 1% (v/v) streptomycin and 10% (v/v)fetal calf serum. After incubation overnight at 37° C., 10 μl/wellnocodazole at a final concentration of 0.1 μg/ml were added to cells.After 24 h incubation, cells were arrested at G2/M phase of the cellcycle progression. Test compounds solubilised in dimethyl sulfoxide(DMSO) were added at various concentrations (0 μM, as well as in therange of 0.005 μM-10 μM; the final concentration of the solvent DMSO was0.5% (v/v)). Cells were incubated for 4 h at 37° C. in the presence oftest compounds. Thereafter, cells were fixed in 4% (v/v)paraformaldehyde in phosphate buffered saline (PBS) at 4° C. overnightthen permeabilised in 0.1% (v/v) Triton X™ 100 in PBS at roomtemperature for 20 min and blocked in 0.5% (v/v) bovine serum albumin(BSA) in PBS at room temperature for 15 min. After washing with PBS, 20μl/well antibody solution (anti-phospho-histone H3 clone 3H10, FITC;Upstate, Cat#16-222; 1:200 dilution) was added to cells, which wereincubated for 2 h at room temperature. Afterwards, cells were washedwith PBS and 20 μl/well HOECHST 33342 dye solution (5 μg/ml) was addedto cells and cells were incubated 12 min at room temperature in thedark. Cells were washed twice with PBS then covered with PBS and storedat 4° C. until analysis. Images were acquired with a Perkin Elmer OPERA™High-Content Analysis reader. Images were analyzed with image analysissoftware MetaXpress™ from Molecular devices utilizing the Cell Cycleapplication module. In this assay both labels HOECHST 33342 andphosphorylated Histone H3 on serine 10 were measured. HOECHST 33342labels DNA and is used to count cell number. The staining ofphosphorylated Histone H3 on serine 10 determines the number of mitoticcells. Inhibition of Mps-1 decreases the number of mitotic cells in thepresence of nocodazole indicating an inappropriate mitotic progression.The raw assay data were further analysed by four parameter logisticregression analysis to determine the IC₅₀ value for each testedcompound.

Investigation of In Vitro Metabolic Stability in Rat Hepatocytes(Including Calculation of Hepatic In Vivo Blood Clearance (CL))

Hepatocytes from Han Wistar rats were isolated via a 2-step perfusionmethod. After perfusion, the liver was carefully removed from the rat:the liver capsule was opened and the hepatocytes were gently shaken outinto a Petri dish with ice-cold WME. The resulting cell suspension wasfiltered through sterile gaze in 50 ml falcon tubes and centrifuged at50×g for 3 min at room temperature. The cell pellet was resuspended in30 ml WME and centrifuged through a Percoll® gradient for 2 times at100×g. The hepatocytes were washed again with Williams' medium E (WME)and resuspended in medium containing 5% FCS. Cell viability wasdetermined by trypan blue exclusion.

For the metabolic stability assay liver cells were distributed in WMEcontaining 5% FCS to glass vials at a density of 1.0×10⁶ vital cells/mL.The test compound was added to a final concentration of 1 μM. Duringincubation, the hepatocyte suspensions were continuously shaken andaliquots were taken at 2, 8, 16, 30, 45 and 90 min, to which equalvolumes of cold methanol were immediately added. Samples were frozen at−20° C. over night, after subsequently centrifuged for 15 minutes at3000 rpm and the supernatant was analyzed with an Agilent 1200HPLC-system with LCMS/MS detection.

The half-life of a test compound was determined from theconcentration-time plot. From the half-life the intrinsic clearanceswere calculated. Together with the additional parameters liver bloodflow, amount of liver cells in vivo and in vitro. The hepatic in vivoblood clearance (CL) and the maximal oral bioavailability (F_(max)) wascalculated. The following parameter values were used: Liver bloodflow—4.2 L/h/kg rat; specific liver weight—32 g/kg rat body weight;liver cells in vivo—1.1×10⁸ cells/g liver, liver cells invitro—0.5×10⁶/ml.

Determination of Inhibitory Potential on Human CYP3A4

The potential of the test compound to act as a competitive inhibitor ofCYP3A4 was evaluated in in vitro assays, using human liver microsomesand the reference substrate midazolam. The test compound was solved inacetonitrile. Human liver microsomal preparation (pool of HLM) wasapplied for the assay. A stock solution of the test compound was addedto phosphate buffer containing EDTA, NADP, glucose 6-phosphate, andglucose 6-phosphate dehydrogenase. This mixture was sequentially dilutedon a Genesis Workstation (Tecan, Crailsheim, FRG). After pre-warming,reaction was initiated by addition of a mixture of probe substrate(midazolam). Finally, the incubation mixtures contained human livermicrosomes at protein concentration of 60 μg/mL, NADPH-regeneratingsystem (1 mM NADP, 5.0 mM glucose 6-phosphate, glucose 6-phosphatedehydrogenase (1.5 U/mL), 1.0 mM EDTA, the test compound at 6 differentconcentrations, 2.5 μM midazolam as probe substrate, and phosphatebuffer (50 mM, pH 7.4) in a total volume of 200 μL. Incubations wereperformed on a Genesis Workstation (Tecan, Crailsheim, FRG) in 96-wellplates (Microtiter plate, 96-well plate) at 37° C. Stock solution ofprobe substrate was prepared in water (midazolam 10 mM). Ketoconazolewas used as positive control of a direct-acting inhibitor. The referencesamples (substrate, but no inhibitor) were incubated in parallel insextuple and contained the same amount of solvent as the testincubations. Reactions were stopped by addition of 100 μL acetonitrilecontaining the internal standard. Precipitated proteins were removed bycentrifugation of the well plate, supernatants were analyzed byLC-MS/MS.

The CYP3A4-mediated metabolic activity in the presence of the testcompounds was expressed as percentages of the corresponding referencevalue. A sigmoid-shaped curve was fitted to the data to calculate theenzyme inhibition parameter IC50 using a nonlinear least-squaresregression analysis of the plot of percent control activity versusconcentration of the test inhibitor. Observing less than 50% inhibition,the data were not extrapolated; hence, IC50 were reported as beinggreater than the highest concentration of the test compound applied.

Determination of Mechanism-Based Inhibition of Human CYP3A4

Mechanism-based (irreversible) inhibition of CYP3A4 was investigated invitro using either human liver microsomes (0.5 mg/mL) or recombinantCYP3A4 isoform (50 or 100 pmol/mL). The test compound was incubated at37° C. in a concentration-NADPH-, and time-dependent setting. Therefore,the test compound was incubated at 4 different concentrations (e.g.: 1,2, 5, 10 μM) in the presence of NADPH (1 M) and regenerating system inphosphate-buffered saline (0.05 mM, pH 7.4) The highest concentration oftest compound was incubated without NADPH and therefore served as theNADPH-deficient control. In addition, acetonitrile as a vehicle control(negative control) and mibefradil as positive control for irreversibleCYP3A4 inactivation were also included using identical assay conditions.

Each mixture was incubated with the enzyme (preincubation) for up to 50minutes. In the beginning and every 10 minutes an aliquot of thisincubation mixture was removed and further diluted by a factor of 10-50(preferred 25) into a new incubation containing NADPH (1 mM) and 150 μMtestosterone as the specific substrate for CYP3A4 (main incubation) andallowed to incubate an additional amount of time (e.g. 10 to 20 min) togenerate the isoform specific metabolite for the final enzyme activityassessment.

Afterwards, the reaction in the main incubation was stopped by theaddition of acetonitrile (containing an internal standard), the proteinwas allowed to precipitate by placing the mixture into an ice bath.Subsequently, the mixture was centrifuged to pelletize the protein andthe supernatants were analyzed by HPLC-MS for the isoform specificmetabolite e.g. 6-13 hydroxytestosterone and the remaining amount of thetest compound.

The remaining enzyme activity was normalized to the initial activity attime 0 min, and then displayed in a semilogarithmic plot dependent onpreincubation time. In parallel the turnover of the substrate wasplotted against the preincubation time. From these data kineticparameters, such as the efficiency of this process expressed by thepartition ratio r (number of inactivator molecules metabolized byprotein molecule of CYP3A4 inactivated) were calculated. For a generaldescription how to determine the partition ratio see R. B. SilvermanMethods Enzymol 1995, 249, 240-283.

The invention claimed is:
 1. A compound of formula (A), (B) or (C):

or a stereoisomer, a tautomer, an N-oxide, a hydrate, a solvate, or asalt thereof, or a mixture of same.
 2. A pharmaceutical compositioncomprising a compound according to claim 1, or a stereoisomer, atautomer, an N-oxide, a hydrate, a solvate, or a salt thereof,particularly a pharmaceutically acceptable salt thereof, or a mixture ofsame, and a pharmaceutically acceptable diluent or carrier.
 3. Apharmaceutical combination comprising: a compound according to claim 1,or a stereoisomer, a tautomer, an N-oxide, a hydrate, a solvate, or apharmaceutically acceptable salt thereof, or a mixture of same; and oneor more agents selected from: a taxane, Docetaxel, Paclitaxel, or Taxol;an epothilone, Ixabepilone, Patupilone, or Sagopilone; Mitoxantrone;Predinisolone; Dexamethasone; Estramustin; Vinblastin; Vincristin;Doxorubicin; Adriamycin; Idarubicin; Daunorubicin; Bleomycin; Etoposide;Cyclophosphamide; Ifosfamide; Procarbazine; Melphalan; 5-Fluorouracil;Capecitabine; Fludarabine; Cytarabine; Ara-C;2-Chloro-2″-deoxyadenosine; Thioguanine; an anti-androgen, Flutamide,Cyproterone acetate, or Bicalutamide; Bortezomib; a platinum derivative,Cisplatin, or Carboplatin; Chlorambucil; Methotrexate; and Rituximab. 4.A method of preparing a compound according to claim 1, comprisingreacting an intermediate compound of formula (A1), (B1) or (C1):

in which Z′ represents a group selected from: —C(═O)OH and—C(═O)O—(C₁-C₆-alkyl); with a compound of formula Ib:

thereby giving, upon optional deprotection, a compound of formula (A),(B) or (C) as defined in claim
 1. 5. A method of preparing a compoundaccording to claim 1, comprising reacting an intermediate compound offormula (A2), (B2) or (C2):

in which Q² is a leaving group; with a compound of formula IIa:

in which Y is a substituent which is displaced in a coupling reaction;thereby giving, upon optional deprotection, a compound of formula (A),(B) or (C), as defined in claim
 1. 6. A method of preparing a compoundaccording to claim 1, comprising reacting an intermediate compound offormula (A3), (B3) or (C3):

in which Q¹ is a leaving group; with a compound of formula (A4), (B4) or(C4):

thereby giving, upon optional deprotection, a compound of formula (A),(B) or (C), as defined in claim
 1. 7. A method of preparing a compoundaccording to claim 1, comprising reacting an intermediate compound offormula (A5), (B5) or (C5):

in which Q³ is an optionally protected NH₂-group; with a compound offormula (A6), (B6) or (C6):

thereby giving, after reduction of the imine, and upon optionaldeprotection, a compound of formula (A), (B) or (C), as defined inclaim
 1. 8. A method of preparing a compound according claim 1,comprising reacting an intermediate compound of formula (A7), (B7) or(C7):

in which R^(3′) is a leaving group; with a compound of formula (A8),(B8) or (C8):

in which Y is a substituent which is displaced in a coupling reaction;thereby giving a compound of formula (A), (B) or (C), as defined inclaim
 1. 9. A compound according to claim 1, which is


10. A compound according to claim 1, which is a salt of